Neurobiology of Epilepsy From Genes To Networks 1St Edition Elsa Rossignol Full Chapter

Neurobiology of Epilepsy From Genes
to Networks 1st Edition Elsa Rossignol

Visit to download the full and correct content document:
https://ebookmass.com/product/neurobiology-of-epilepsy-from-genes-to-networks-1st-
edition-elsa-rossignol/
Serial Editor
Vincent Walsh
Institute of Cognitive Neuroscience
University College London
17 Queen Square
London WC1N 3AR UK
Editorial Board
Mark Bear, Cambridge, USA.

Medicine & Translational Neuroscience
Hamed Ekhtiari, Tehran, Iran.
Addiction
Hajime Hirase, Wako, Japan.
Neuronal Microcircuitry
Freda Miller, Toronto, Canada.
Developmental Neurobiology
Shane O’Mara, Dublin, Ireland.
Systems Neuroscience
Susan Rossell, Swinburne, Australia.
Clinical Psychology & Neuropsychiatry
Nathalie Rouach, Paris, France.
Neuroglia
Barbara Sahakian, Cambridge, UK.
Cognition & Neuroethics
Bettina Studer, Dusseldorf, Germany.
Neurorehabilitation
Xiao-Jing Wang, New York, USA.
Computational Neuroscience
Elsevier
Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK
50 Hampshire Street, 5th Floor, Cambridge, MA 02139, USA
First edition 2016
Copyright # 2016 Elsevier B.V. All rights reserved
No part of this publication may be reproduced or transmitted in any form or by any means,
electronic or mechanical, including photocopying, recording, or any information storage and
retrieval system, without permission in writing from the publisher. Details on how to seek
permission, further information about the Publisher’s permissions policies and our
arrangements with organizations such as the Copyright Clearance Center and the Copyright
Licensing Agency, can be found at our website: www.elsevier.com/permissions.
This book and the individual contributions contained in it are protected under copyright by the
Publisher (other than as may be noted herein).
Notices
Knowledge and best practice in this field are constantly changing. As new research and
experience broaden our understanding, changes in research methods, professional practices, or
medical treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in
evaluating and using any information, methods, compounds, or experiments described herein.
In using such information or methods they should be mindful of their own safety and the safety
of others, including parties for whom they have a professional responsibility.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors,
assume any liability for any injury and/or damage to persons or property as a matter of products
liability, negligence or otherwise, or from any use or operation of any methods, products,
instructions, or ideas contained in the material herein.
ISBN: 978-0-12-803886-4
ISSN: 0079-6123
For information on all Elsevier publications

visit our website at https://www.elsevier.com/
Publisher: Zoe Kruze

Acquisition Editor: Kirsten Shankland
Editorial Project Manager: Hannah Colford
Production Project Manager: Magesh Kumar Mahalingam
Cover Designer: Greg Harris
Typeset by SPi Global, India
Contributors
A. Alexander
Stanford University, Stanford, CA, United States
S.C. Baraban
Epilepsy Research Laboratory, University of California, San Francisco, CA, United
States
S. Baulac
Sorbonne Universit es, UPMC Univ Paris 06, UM 75; INSERM, U1127; CNRS,
UMR 7225; ICM (Institut du Cerveau et de la Moelle epinière); AP-HP Groupe
hospitalier Piti
e-Salp^
etrière, Paris, France
D.A. Coulter
Perelman School of Medicine, University of Pennsylvania; The Research Institute
of the Children’s Hospital of Philadelphia, Philadelphia, PA, United States
C.G. Dengler
Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA,
United States
J. Ghaziri
Research Centre, Centre hospitalier de l’Universite de Montreal, Montreal, QC,
Canada
A. Griffin
States
F. Gu
Epilepsy Research Laboratories, Stanford University School of Medicine,
Stanford, CA, United States
A.E. Hernan
University of Vermont College of Medicine, Burlington, VT, United States
G.L. Holmes
University of Vermont College of Medicine, Burlington, VT, United States
X. Jiang
Universit
e de Montr
eal; CHU Ste-Justine Research Center, Montreal, QC, Canada
C. Krasniak
States
M. Lachance
CHU Ste-Justine Research Center, Montreal, QC, Canada
M. Maroso
v
vi Contributors
H.C. Mefford
University of Washington, Seattle, WA, United States
C.T. Myers
D.K. Nguyen
Research Centre, Centre hospitalier de l’Universite de Montreal; CHUM–H^opital
Notre-Dame, Montreal, QC, Canada
I. Parada
D.A. Prince
E. Rossignol
Universit
e de Montr
eal; CHU Ste-Justine Research Center, Montreal, QC, Canada
I. Soltesz
Y. Zerouali
Research Centre, Centre hospitalier de l’Universite de Montreal; Ecole
Polytechnique de Montr
eal, Montreal, QC, Canada
Preface
The following volume stems from a meeting of the same name “The Neurobiology of
Epilepsy: From Genes to Networks” held in Montreal on May 4–5, 2015, and orga-
nized by Drs. L. Carmant, P. Cossette, E. Rossignol, and J.-C. Lacaille. The editors
would like to acknowledge the support of the Groupe de Recherche sur le Système
Nerveux Central, Universite de Montreal, for the organization of the meeting.
Epilepsy is a brain disease caused by abnormal and excessive electrical discharges
of neurons. The underlying etiologies are multiple, but recent research indicates an
important role for pathological genetic variants causing dysregulation of neuronal
networks. This meeting brought together an international group of clinicians and
basic scientists to share new information on the neurobiological basis of epilepsy,
including clinical aspects, molecular mechanisms, neuronal networks, as well as
animal models and novel therapies. By trying to discuss the “neurobiology” of
epilepsy, we mean to address the fundamental mechanisms underlying the genetic
basis of epilepsy and hopefully lead to an understanding of epilepsy at the molecular,
cellular, and network levels that will be translatable into improved treatment for
patients with epilepsy.
The volume begins with sections covering novelties in the clinical investigation
of patients with epilepsy. Drs. Zerouali, Ghaziri, and Nguyen describe multimodal
imaging techniques involved in the investigation of epileptic networks in patients,
focusing on insular cortex epilepsy. Drs. Myers and Mefford review current knowl-
edge on the genetic investigation techniques used to identify molecular etiologies
in patients with epileptic encephalopathies, and provide an overview of the clinical
features and basic mechanisms of recently described genetic epileptic encephalop-
athies. Dr. Baulac examines how germline and somatic mutations in the genes of the
GATOR1 complex, which regulates the mTOR pathway, cause focal epilepsies with
variable foci.
An understanding of the “neurobiology” of epilepsy must also elucidate seizures
at the microcircuit level and understand how neuronal networks are affected in ep-
ilepsy. The volume continues with three chapters discussing the molecular, cellular,
and network mechanisms involved in the genetics of epilepsy. Drs. Jiang, Lachance,
and Rossignol consider the involvement of cortical GABAergic interneuron disor-
ders in genetic causes of epilepsy; Drs. Alexander, Maroso, and Soltesz discuss work
on the organization and control of epileptic circuits in temporal lobe epilepsy; and
Drs. Dengler and Coulter review the normal and epilepsy-associated pathologic
function of the hippocampal dentate gyrus.
A major justification for elucidating the genetic, molecular, cellular, and network
basis of epilepsies is to develop effective treatment therapies for patients. The vol-
ume then moves into investigations of animal models and therapies. Drs. Hernan and
Holmes examine work on antiepileptic treatment strategies in neonatal epilepsy, and
Drs. Griffin, Krasniak, and Baraban discuss advancement in epilepsy treatment
through personalized genetic zebrafish models.
xi
xii Preface
Finally, the concluding chapter for the volume is from Drs. Prince, Gu, and
Parada, describing antiepileptogenic repair of excitatory and inhibitory synaptic
connectivity after neocortical trauma.
Elsa Rossignol, Lionel Carmant, and Jean-Claude Lacaille

Departement de neurosciences, Universite de Montreal, Montreal, Canada
CHAPTER
Multimodal investigation of
epileptic networks: The case
of insular cortex epilepsy
Y. Zerouali*,†, J. Ghaziri*, D.K. Nguyen*,{,1
1
*Research Centre, Centre hospitalier de l’Universit
e de Montr
eal, Montreal, QC, Canada
†
Ecole Polytechnique de Montr eal, Montreal, QC, Canada
{
CHUM–H^ opital Notre-Dame, Montreal, QC, Canada
1
Corresponding author: Tel.: +1-514-890-8000 ext. 25070; Fax: +1-514-338-2694,
e-mail address: d.nguyen@umontreal.ca
Abstract
The insula is a deep cortical structure sharing extensive synaptic connections with a variety of
brain regions, including several frontal, temporal, and parietal structures. The identification of
the insular connectivity network is obviously valuable for understanding a number of cognitive
processes, but also for understanding epilepsy since insular seizures involve a number of re-
mote brain regions. Ultimately, knowledge of the structure and causal relationships within the
epileptic networks associated with insular cortex epilepsy can offer deeper insights into this
relatively neglected type of epilepsy enabling the refining of the clinical approach in managing
patients affected by it.
In the present chapter, we first review the multimodal noninvasive tests performed during
the presurgical evaluation of epileptic patients with drug refractory focal epilepsy, with par-
ticular emphasis on their value for the detection of insular cortex epilepsy.
Second, we review the emerging multimodal investigation techniques in the field of epi-
lepsy, that aim to (1) enhance the detection of insular cortex epilepsy and (2) unveil the archi-
tecture and causal relationships within epileptic networks. We summarize the results of these
approaches with emphasis on the specific case of insular cortex epilepsy.
Keywords
Epilepsy, Insula, Connectivity, Networks, Multimodal, Causality, Neuroimaging
1 EPILEPSY AS A SYSTEMS DISEASE

For most epileptic patients (70%), anticonvulsive drugs adequately control sei-
zures. However, among the refractory cases, a significant proportion of patients
are eligible for surgical treatment of seizures (Wiebe et al., 2001). The fundamental
Progress in Brain Research, Volume 226, ISSN 0079-6123, http://dx.doi.org/10.1016/bs.pbr.2016.04.004
© 2016 Elsevier B.V. All rights reserved.
1
2 CHAPTER 1 Insular cortex epileptic networks
question in those cases is to localize the part of the brain that is responsible for pa-
tients’ seizures, which constitutes the central thread of this chapter. Important ad-
vances in the surgical treatment of epilepsy arose from both a better formulation
of this question and the development of methodological tools to answer it. Indeed,
our notion of epilepsy has evolved from a local-based to network-based model, cap-
italizing on the ability of neuroimaging to study brain function at increasingly high
spatial and temporal resolutions.
Early in the last century, the measurement of brain electrical potentials on the
scalp by Berger paved the way for the investigation of the neuroelectric correlates
of epileptic seizures. In addition to seizures, Berger also reported the existence of
sharp electrical transients that are observable on the electroencephalogram (EEG)
of epileptic patients in the absence of seizures. These “spikes” are usually observed
on electrodes that record seizures but this is not always the case. This spatial distinc-
tion between the generators of seizures and spikes was further elaborated with the
advent of intracranial EEG recordings (icEEG). icEEG allows excellent spatial dis-
crimination of the neural generators of epileptic activity, which led Laufs and Rose-
now to propose a “zonal” model to explain the pathophysiology of epilepsy
(Rosenow, 2001). The “zonal” model recognizes different zones associated with
the clinical symptoms (symptomatogenic zone), the interictal spikes (irritative zone),
the initiation of seizures (seizure onset zone—SOZ), and the functional deficits as-
sociated with the epileptic condition (functional deficit zone). Importantly, they de-
fine an “epileptogenic” zone (EZ) that consists of the brain tissue that must be
surgically resected for seizures to be cured. The spatial location of the EZ is usually
estimated using multimodal investigation techniques, as will be described in the
next section, but its true location can only be confirmed through positive surgical
outcome.
Although the zonal concept of epilepsy had an important impact on the clinical
management of epileptic patients, failure rates for epilepsy surgeries remain rela-
tively important, as high as 30% for temporal lobe—TLE (Jeha et al., 2006;
Wiebe et al., 2001) and 50% for frontal lobe—FLE (Jeha et al., 2007; Yun et al.,
2006) and parietal lobe—PLE (Binder et al., 2009; Kim et al., 2004a) epilepsy. In
2002, Spencer formulated the idea that we should envision generators of interictal
and ictal activities as networks of structures rather than single zones (Spencer,
2002). Since the transition from interictal to ictal to postictal brain states occurs
at the time scale of synaptic activity, this idea has two corollaries. First, it implies
that the neural machinery supporting the emergence of epileptogenic networks
(ENs) is hardwired into the brain (Richardson, 2012). Thus, epilepsy is a systems
disease, the symptoms of which result from aberrant connectivity among a set of
anatomical healthy structures (Avanzini and Franceschetti, 2003). Some authors sug-
gest that neural networks are bistable systems that can exhibit both healthy and ep-
ileptiform activity for the same set of parameters (Breakspear et al., 2006; Da Silva
et al., 2003; Marten et al., 2009). Dynamical transitions between these two states are
called bifurcations, and the epileptic condition facilitates such bifurcations.
The second corollary is that the network assembly is a highly flexible process; for
a given set of components, there are a large number of network architectures, all of
2 Investigating the epileptic networks: Clinical practice 3
which may give rise to different epileptiform activities and clinical symptoms. This
idea has deep implications for clinicians and neuroscientists, since accurate locali-
zation of network components is insufficient for fully describing the pathophysiol-
ogy of epilepsy. For such an endeavor, it is necessary to study time-varying network
dynamics (Halász, 2010).
In order to illustrate the networks concept of epilepsy and the current techniques
used in their investigation, we use the unique case of insular cortex epilepsy (ICE).
The insula is a cortical structure located deep in the sylvian fissure and is hidden by
the frontal, temporal, and parietal opercula. Despite early reports on insular epilep-
tiform activity (Guillaume and Mazars, 1949; Penfield and Faulk, 1955; Penfield and
Jasper, 1954), insulectomy was not considered an efficient surgical approach
(Silfvenius et al., 1964) until the past 15 years. Patient series from Isnard et al.
(2004) and Ryvlin and Kahane (2005) demonstrated that insular ENs include tem-
poral, frontal, and parietal structures and that the sequence of clinical symptoms as-
sociated with insular seizures can be explained by their patterns of propagation.
Throughout this chapter, we will present multimodal investigation techniques used
both for localizing the components of ENs and characterizing their architectures,
with emphasis on ICE. Section 2 presents the investigation techniques that are rou-
tinely used in most epilepsy centers for imaging the epileptogenic brain tissues.
Section 3 presents new experimental investigation techniques that promise enhanced
imaging and understanding of ENs.
2 INVESTIGATING THE EPILEPTIC NETWORKS: CLINICAL

PRACTICE
2.1 PRESURGICAL INVESTIGATION TECHNIQUES
2.1.1 icEEG
The gold standard in the localization of the anatomical components of epileptogenic
networks consists of direct recordings of neuroelectric activity through electrodes in
contact with brain tissue. icEEG records local field potentials that are generated by
neural populations within a 0.5–3 mm radius from the tip of the electrode (Juergens
et al., 1999; Mitzdorf, 1987); thus, achieving the highest spatial resolution among
all neuroimaging techniques used in clinic. The downside of such high resolution
is obviously poor spatial coverage, since only a limited number of electrodes can
be used without risking cerebral hemorrhage or neurological deficits (Wong et al.,
2009). It is thus possible that the epileptogenic zone is not sampled by icEEG,
leading to the wrong selection of surgical target. ICE provides an ideal illustration
of this issue.
Insufficient insular coverage in patients with ICE was associated with a significant
proportion of failed TLE, FLE, and PLE surgeries. Initially, suspicions were raised
by a study on patients with TLE with atypical clinical symptoms that were instead as-
sociated with insular activity (Aghakhani et al., 2004). Despite successive resections
(up to four) of anterior temporal, mesiotemporal, and parietotemporal structures,
patients continued having seizures. Unfortunately, no electrode sampled the insula in

their study although insular hyperperfusion was clearly shown in one patient. The
potential benefit from icEEG recordings in the insula in TLE was further demon-
strated, as about 10% of patients diagnosed with TLE suffered from ICE (Isnard
et al., 2004). TLE-like symptoms in those patients were explained by secondary prop-
agation of ictal activity to surrounding temporal structures. Similar conclusions were
drawn by some studies on PLE and FLE (Roper et al., 1993; Ryvlin et al., 2006).
Based on these reports, our group lowered its decision threshold for insular im-
plantations with depth electrodes in patients with nonlesional TLE, FLE, and PLE.
On a series of 18 patients meeting these criteria, we found that 40% patients who
underwent icEEG recordings had seizures originating from the insula. In addition,
electrical stimulation of the insula proved that insular epileptic discharges replicate
semiology of various extrainsular epilepsies (Nguyen et al., 2009). Our findings,
along with existing literature on this issue suggest that (1) ICE is probably more prev-
alent than presently reported; more extensive studies must be conducted to determine
its frequency, (2) despite extensive presurgical workups, nonlesional ICE is probably
rarely detected, accounting for a significant proportion of failed epilepsy surgeries.
We further review the different investigation techniques used in presurgical workups
and discuss their value for detecting ICE.
2.1.2 EEG
EEG is probably the oldest and most widely used imaging modality in clinical
investigations of brain pathologies, including epilepsy. Over the years, epileptolo-
gists developed expert skills in reading and interpreting EEG seizures, but also
waveforms observed during the interictal state, such as spikes, polyspikes, spike-
and-wave complexes, sharp waves, paroxysmal fast activity (Westmoreland, 1998),
and high-frequency oscillations (Bragin et al., 1999). Advanced biophysical models
and computerized techniques allow unprecedented accuracy in the localization of those
waveforms, as most advanced algorithms can theoretically reach a 10 cm2 resolution
(Grova et al., 2006), thus enabling “electrical source imaging” (ESI).
ESI relies on a biophysical model that relates neural electrical activity, modeled
as a finite number of equivalent current dipoles (ECD), to electrical potentials
recorded outside the head. We distinguish two broad classes of ESI techniques,
according to the number ECD used for modeling brain activity. Single ECDs as-
sume recorded electrical potentials are generated by a single (or a few) neural point
source(s). Although this approach obviously oversimplifies neural generators and
its numerical implementation requires strong heuristics (number of dipoles, initial-
ization), it proved valuable in epilepsy when careful attention is paid to its limita-
tions, as validated by simultaneous EEG and icEEG recordings (Boon et al., 2002;
Ebersole, 1991; Roth et al., 1997). In general, all studies report usefulness of sECD
for epilepsy, with sensitivity and specificity exceeding, respectively, 80% and 60%
for the vast majority of studies (see Kaiboriboon et al., 2012, for a review).
In turn, distributed source modeling (DSM) models neural sources with a large
number of ECDs homogenously distributed in the brain. Despite this mathematical
challenge imposed by the large number of sources, DSM is increasingly being
validated in the clinical management of epileptic patients, benefiting from increased

head coverage of high-density EEG systems and increased accuracy of head model-
ing techniques. In the study with the largest cohort of patients, DSM was shown to
accurately localize the onset of seizures, with sensitivity and specificity both exceed-
ing 80% (Brodbeck et al., 2011), in line with several other reports (Michel et al.,
2004; Sperli et al., 2006). In ICE, EEG was found to be insensitive to spikes gener-
ated from the deep-seated insula; therefore, we did not find any reports on the use-
fulness of ESI for that kind of epilepsy.
2.1.3 MEG
Magnetoencephalography (MEG) is a relatively new imaging modality that records
the magnetic fields generated by electrical currents flowing inside active neurons.
Despite a relatively short history of clinical investigation and higher operation costs
than EEG, MEG has quickly established itself as an important tool in presurgical
evaluation of epilepsy. Owing to the relatively simpler physics of neural magnetic
fields propagation, MEG is sensitive to smaller activated brain regions (4 cm2
for MEG, Mikuni et al., 1997, 10 cm2 for EEG, Grova et al., 2006). Source local-
ization with DSM showed promising results for the presurgical workup of epilepsy,
which led some authors to suggest that it could obviate icEEG investigations in some
cases (Fujiwara et al., 2012).
Several studies demonstrated the usefulness of sECD modeling with MEG on
different types of epilepsy. Globally, the reported accuracy of sECD is above
75% for the majority of the studies (Knowlton, 2006; Minassian et al., 1999;
Stefan, 1993). In addition, MEG was also shown to be valuable for appropriately
determining the subsequent icEEG coverage zone (Fischer et al., 2005;
Knowlton et al., 2009; Mamelak et al., 2002). Unfortunately, only a few studies
report sECD investigation of ICE. Among those, Heers et al. studied three patients
with cryptogenic epilepsy and hypermotor seizures (Heers et al., 2012). They
showed that MEG can not only identify epileptic foci when other modalities fail
but is also able to localize deep-seated foci such as in the insula (Park et al.,
2012). We recently investigated 14 patients with insular seizures using MEG.
Among those, localization of interictal spikes showed clear insular or perisylvian
focus in all but one patient. Taken together, these studies suggest that MEG is valu-
able for detecting ICE.
In turn, we found substantially fewer reports of DSM, probably due to the fact it is
more recent and less validated. Nonetheless, studies using DSM advocate for its
more extensive use in presurgical workups since it outperformed sECD in at least
two comparative studies (Shiraishi et al., 2005; Tanaka et al., 2009). It was also
shown that patterns of source activity reconstructed with DSM were good predictors
(up to 94%) for subsequent surgical resection (Tanaka et al., 2010).
2.1.4 MRI
Magnetic resonance (MR) scanners exploit the intrinsic magnetic properties (the
spin) of electrons to produce high-resolution images of brain (and body) tissues.
Using a sophisticated combination of spin polarization and perturbation, MR
imaging (MRI) now allows recovering the three-dimensional properties of the brain
with 1 mm resolution. MRI is an essential tool in the evaluation of patients with focal
epilepsy, allowing the visual detection of a variety of epileptogenic brain lesions
such as gliosis from acquired insults, tumors, vascular malformations, or malforma-
tions of cortical development. Globally, the sensitivity of MRI in epilepsy surgery
candidates ranges between 75% and 86% (Bronen et al., 1996; Brooks et al.,
1990; Cascino et al., 1992; Grattan-Smith et al., 1993; Kuzniecky et al., 1993;
Laster et al., 1985; Latack et al., 1986; Ormson et al., 1986; Scott et al., 1999).
Literature on ICE provides contrasting results with respect to the sensitivity of
MRI. We found six studies in which all patients (N ¼ 43) displayed some abnormality
(Cukiert et al., 1998; Duffau et al., 2002; Heers et al., 2012; Kaido et al., 2006; Roper
et al., 1993; von Lehe et al., 2008), two studies (N ¼ 23) with both MRI-positive and
MRI-negative patients (Malak et al., 2009; Mohamed et al., 2013), and seven studies
(N ¼ 19) with exclusively MRI-negative patients (Dobesberger et al., 2008; Isnard
et al., 2000, 2004; Kriegel et al., 2012; Nguyen et al., 2009; Ryvlin et al., 2006;
Zhang et al., 2008). Overall, MRI had a sensitivity of 61%, corresponding to 87%
positive predictive value when considering the results of surgery. Overall, our short
review suggests that MRI is a highly valuable tool for investigating ICE with mod-
erate sensitivity but excellent diagnostic value. However, the relatively larger num-
ber of patients diagnosed with lesional rather than nonlesional ICE might reflect the
fact that nonlesional ICE is a poorly diagnosed disease, and that currently available
investigation tools other than MRI have low diagnostic value for this disease.
2.1.5 PET
Positron-emission tomography (PET) measures the concentration of a specific
source of radiation in the 3D space. The brain property imaged with PET thus
depends on the choice of an appropriate radioligand, such that 3D concentrations
can be interpreted in terms of brain function. In epilepsy, 2-[18F]-fluoro-2-
deoxy-D-glucose (FDG) and [11C]-flumazenil (FLU) are two complimentary and
commonly used radioligands, imaging brain function (glucose consumption), and
structure (neuronal loss), respectively. The epileptic condition is associated with
neuronal loss and, paradoxically, decreased concentrations of FDG in affected brain
regions. Although this last observation is largely consensual, the underlying neuro-
physiological mechanisms are still not understood.
Studies comparing FLU- and FDG-PET for localizing the brain regions involved
in epileptogenic networks report similar performance of both molecules for most
groups of patients. Globally, these two modalities achieve similar sensitivity and
specificity (Ryvlin et al., 1998) and are equally predictive with respect to surgical
outcome (Debets et al., 1997). It was noted however that the only cases where
FLU-PET added new information above that of MRIs are when FDG-PET is nega-
tive, which implies that FDG-PET should be performed first, and FLU-PET only
when FDG-PET is negative.
Since the role of insula has only gained recognition in the last 10 years, early PET
studies reporting changes in metabolism in the insula did not include insular intra-
cranial electrodes (Bouilleret et al., 2002; Didelot et al., 2008; Hur et al., 2013; Joo,
2005; Wong et al., 2010). For this reason, although they provided interesting insights
into the involvement of the insula in the metabolic changes associated with TLE,
they are inconclusive with regard to ICE. We found few reports of ICE with icEEG
and PET data (Dobesberger et al., 2008; Heers et al., 2012), including two studies
from our group (Nguyen et al., 2009; Surbeck et al., 2014). Pooled together, these
studies suggest that FDG-PET has low sensitivity (17%) and specificity (53%)
to ICE.
2.1.6 SPECT
Single-photon emission computerized tomography (SPECT) consists in localizing
the source of gamma-ray emission within the brain. The source consists of a radio-
active tracer (usually the 99mTc-labeled HMPAO—hexamethylpropyleneamine ox-
ime) bound to a molecule that freely crosses the blood-brain barrier, such that it
diffuses into the brain after intravenous injection. Since the tracers used in SPECT
have relatively long half-lives, they distribute spatially in brain regions with higher
blood flow and remain stable for up to a few hours. Patients can thus be EEG-video
monitored and injected at the time of a relevant brain activity (ideally seizure onset)
as seen on the EEG traces. It is generally agreed that the use of SPECT during inter-
ictal brain state is of limited use (Debets et al., 1997; Lascano et al., 2015; Spencer,
1994). In contrast, SPECT is mostly useful when the radioligand is injected at seizure
initiation (Joo et al., 2004; Spencer, 1994). In addition, even better results can be
achieved by subtracting interictal from ictal SPECT images, a technique called SIS-
COM. Such an operation is especially useful in cases where ictal hyperperfusion is
low in epileptogenic zone due to superposition on a preceding hypoperfused state
(Desai et al., 2013; Newey et al., 2013; Spencer, 1994; von Oertzen et al., 2011).
As for PET, there are only a few studies that reported on the value of SPECT in
ICE. We found few reports of ICE with icEEG and PET data (Dobesberger et al.,
2008; Heers et al., 2012), including two studies from our group (Nguyen et al.,
2009; Surbeck et al., 2014). Pooled together, these studies suggest that SPECT
has low sensitivity (23%) and specificity (48%) to ICE.
2.2 ILLUSTRATIVE CASE

A 10-year-old ambidextrous girl with mild language delay started having seizures at
the age of 4 years characterized by an unpleasant tingling sensation in the lower back,
right arm, and both legs followed by fear and complex motor behaviors. Seizures
became predominantly nocturnal after a few weeks and have remained so since, re-
curring in clusters of 4 or 5 (up to 15) every 2–3 days. After failing six adequate anti-
epileptic drug trials, the patient was referred for epilepsy surgery. While the clinical
history might have suggested a frontal lobe focus, video-EEG monitoring revealed
right temporal, central, or centro-temporal discharges interictally and right centro-
temporal rhythmic activity at seizure onset (Fig. 1). High-resolution 3T brain
MRI and volumetric studies failed to disclose an epileptogenic lesion. Interictal
FDG-PET was normal but ictal SPECT showed increased cerebral blood flow over
the right insular region (Fig. 2). Source localization of interictal epileptiform
FIG. 1
EEG recordings of the epileptic activity from the illustrative patient. The time axis (horizontal)
is discontinuous as shown by the double vertical black lines. Interictal and ictal activity
are displayed, respectively, to the left and right of the black lines. A clear temporo-central
spike is displayed between the two vertical orange (light gray in the print version) lines.
A seizure starts right after the vertical red (gray in the print version) bar. From those traces,
EEG does not allow the detection of the insular focus.
FIG. 2
Ictal SPECT images from the illustrative patient. The selected coronal slices are displayed
in the neurological convention (right hemisphere at the right) and span the antero-posterior
axis of the insula. These slice show clear asymmetry in blood perfusion, the right insula
showing clear hyperperfusion as compared to the left side.
discharges recorded during MEG (CTF 275-sensory system, Canada) using an elec-
trical current dipole model revealed a concordant tight cluster at the very posterior
end of the right Sylvian fissure (posterior insula and parietal more than temporal
opercula—Fig. 3). During spikes, combined EEG–fMRI recordings showed (blood
oxygen level-dependent, BOLD) activations over the right posterior insula, the over-
lying perisylvian cortex but also in central regions and the cingulate gyrus (Fig. 4).
Functional MRI for language suggested left-hemisphere dominance. Based on this
multimodal noninvasive evaluation, epilepsy surgery was recommended. With the
removal of the right parietal operculum, temporal operculum, and posterior insula,
seizure-freedom was attained (follow-up 2 years).
FIG. 3
Single-dipole modeling of the interictal spikes recorded from the illustrative patient using
MEG, each dipole corresponding to a single spike. Localized dipoles clearly cluster in the
posterior portion of the insula and in the centro-parietal opercula.
FIG. 4
CombinedEEG–fMRI recordings of interictal spikes from one patient of our cohort of
insular cortex epilepsy. General linear model reveals a single BOLD activation cluster in the
posterior portion of the right insula along with the overlying central operculum. Although they
were found in other patients, activation of other structures, such as the cingulate gyrus did not
reach significance.
3 INVESTIGATING THE EPILEPTIC NETWORKS:

PERSPECTIVES
3.1 ADVANCED LOCALIZATION TECHNIQUES
3.1.1 EEG/MEG fusion
Both EEG and MEG directly address neuronal activity by recording differences in
electrical potentials and magnetic fields, respectively, outside the head. Although
both modalities record activity from the whole brain, they are preferentially sensitive
to different populations of neurons. Indeed, EEG is mainly sensitive to radially ori-
ented sources lying on cortical gyri since they are closer to the sensors while the mag-
netic fields generated by those sources vanish due to the quasi-spherical head shape.
In turn, MEG is mainly sensitive to tangential source lying along the sulci walls
(Ahlfors et al., 2010; Sharon et al., 2007). Thus a cortical region participating in
an EN would only be partially recovered by EEG and MEG if it extends spatially
from the top of a gyrus to a cortical fold. Some studies further observed noticeable
epileptogenic activity on one modality but not the other (Barkley and Baumgartner,
2003; Iwasaki et al., 2005), highlighting the need for simultaneous EEG–MEG re-
cordings. Importantly, it was shown that EEG and MEG acquired simultaneously
are superadditive, ie, they provide more information relevant to source localization
than the sum of unimodal information (Pflieger et al., 2000).
Simultaneous EEG/MEG (MEEG) recordings were introduced in epilepsy with
the aim to better delineate the location and spatial extent of cortical sources partic-
ipating in epileptogenic networks. Using simulations of realistic epileptic spikes,
Chowdhury et al. showed that MEEG provides better localization than EEG or
MEG alone, regardless of the inversion scheme used (Chowdhury et al., 2015). How-
ever, contrary to what is commonly believed they showed that the maximum entropy
on the mean framework (Amblard et al., 2004) not only provides the most accurate
localization of the simulated sources but is also able to recover their spatial extent
(Chowdhury et al., 2013; Ebersole and Ebersole, 2010). In addition, in the same
study on two patients with frontal lobe epilepsy, MEEG was able to track interictal
spike propagation patterns while individual modalities were insensitive to spatiotem-
poral dynamics of the spikes. Similar conclusions were drawn from a recent study
comparing unimodal and multimodal MEEG source localizations with intracranially
recorded EEG on a patient with multifocal refractory epilepsy. The authors show that
MEEG is able to recover most of the regions participating in the generation of spikes,
even when no single modality was able to recover them (Aydin et al., 2015). This
study illustrates the supraadditive nature of MEEG and its potential to recover more
components of ENs.
We recently started exploring the potential of MEEG source imaging of epileptic
spikes to detect insular activations in ICE. Extending the previously cited reports, we
found that EEG and MEG are each able to detect insular activations on subsets but
not all spikes. However, MEEG source imaging provided more robust results and
could detect insular activations in cases where only a single modality was positive
(see Fig. 5A) and even when both modalities were negative (Fig. 5B). These
3 Investigating the epileptic networks: Perspectives 11
FIG. 5
Combined EEG–MEG source reconstruction of two epileptic spikes from one patient with ICE.
(A) For this spike, the EEG was unable to detect the insular activation and reveals mainly
activity in the orbitofrontal cortex. In turn, both MEG and MEEG could detect activation in the
ventral region of the insula. (B) For this spike, neither EEG nor MEG could detect insular
activation while MEEG clearly displays maxima of power in the ventral and posterior insular
regions.
preliminary results suggest that MEEG source imaging of epileptic spikes is a prom-
ising avenue for the computer-assisted detection of ICE.
3.1.2 Combined EEG and fMRI

A clinical-grade MR magnet is probably among the most hostile environments for
recording scalp potentials with EEG. Indeed, even small movements of the EEG
electrodes inside the scanner as a result of small head movements or
ballistocardiographic effects, translate into current induction in electrodes. In addi-

tion, the on/off switching of the radiofrequency antennas creates even larger artifacts
on the EEG, two orders-of-magnitude larger than the activity of interest. Nonethe-
less, modern signal processing techniques allow for a proper cleaning of EEG data
such that EEG–fMRI can be used for relating hemodynamic and neuroelectric brain
activity. Given the deterministic nature of the gradient artifact, waveform averaging
was introduced (Allen et al., 2000) and validated (Gonçalves et al., 2007; Salek-
Haddadi et al., 2002) to subtract the artifact from the EEG. Other filtering techniques,
applicable to the ballistocardiographic effect, were also proposed based on spectral
domain filtering (Sijbers et al., 1999), wavelet filtering (Kim et al., 2004b), spatial
Laplacian filtering, PCA (Niazy et al., 2005), and ICA (Mantini et al., 2007;
Srivastava et al., 2005).
In the context of epilepsy, one of the most widely used EEG–fMRI paradigms is
to analyze the BOLD signal in an event-related design where the events are EEG-
marked interictal spikes. After preprocessing, spikes are marked on the EEG by
expert epileptologists. The EEG signal is then binarized according to the spike mark-
ing, downsampled to match fMRI time resolution, and convolved with a model of
the hemodynamic response function that is then cast as a regressor in the general
linear model. The most common model is the canonical hemodynamic response
function (HRF), which accounts for local elastic deformation of blood capillaries
and the resultant transient increase in local blood oxygenation in response to neu-
ronal activity—also termed neurovascular coupling (Buxton et al., 1998). However,
it was shown that the shape and onset of the HR can vary significantly among sub-
jects (Aguirre et al., 1998; Lindquist et al., 2009), with respect to: subjects age
(D’Esposito et al., 1999; Jacobs et al., 2008), brain regions (Handwerker et al.,
2004) and brain lesions including epileptogenic lesions (Lemieux et al., 2008;
Masterton et al., 2010). Several alternate models of the HRF were proposed to ac-
count for such variability, including general nonlinear fits of the HRF, multiple
HRFs with varying onset and peak times, HRF along with its time derivatives
(Friston et al., 1998), general basis functions sets (Josephs and Henson, 1999),
and the superposition of three inverse logit functions (Lindquist et al., 2009). These
models are then integrated in a general linear model to infer the response of each
voxel to the epileptic activity (Friston, 1995). Irrespective of the chosen model, use-
fulness of EEG–fMRI to imaging epileptic networks has been demonstrated in sev-
eral studies.
First, it was shown that the BOLD signal provides useful information for confirm-
ing the location of the suspected epileptic focus. Depending on the study, proportions
of patients who display IED-related changes in the BOLD signal ranged from 67% to
83% (Kobayashi et al., 2006; Salek-Haddadi et al., 2006). The most clinically useful
BOLD changes are activations, since identification of a single activation cluster was
found to be concordant with electro-clinical symptoms in over 80% of cases
(Krakow, 1999; Salek-Haddadi et al., 2006; Thornton et al., 2010) and are good pre-
dictors of positive surgical outcome (An et al., 2013). Importantly, a number of stud-
ies showed that EEG–fMRI has the potential to reveal the components of ENs.
Indeed, in mesial TLE, BOLD occasionally displays significant activation clusters
in the contralateral temporal and extratemporal regions (Avesani et al., 2014;

Kobayashi et al., 2006; Tousseyn et al., 2014). Those clusters were considered as
patterns of spike propagation since surgical outcomes are good despite sparing those
clusters. Similarly, in patients with intractable generalized epilepsy whose interictal
activity is characterized by sharp spike-wave bursts, BOLD changes show significant
activation of the thalamus while EEG does not (Aghakhani, 2004).
We recently conducted an EEG–fMRI study aimed at revealing the EN associated
with ICE (unpublished results). We recruited 13 ICE patients, as confirmed by sur-
gical outcome after insulectomy. We were able to detect IEDs in 62% of patients,
similarly to what has been reported in studies on other kinds of epilepsies. We found
ipsilateral insular and or perisylvian BOLD activations in six patients while the
remaining two displayed significant BOLD activation in the contralateral insula
(data from one patient is shown in Fig. 4). In addition to insular and perisylvian ac-
tivations, significant BOLD clusters were found in the postcentral gyrus, superior
parietal lobule, middle or superior frontal gyri, and anterior cingulate or medial fron-
tal gyri, all of which were previously shown to share structural connectivity with the
insula (Augustine, 1996; Nieuwenhuys, 2012). We thus think that EEG–fMRI is a
promising tool for revealing complex ENs in ICE.
3.1.3 Quantified icEEG

In principle, icEEG recordings are the gold standard in epilepsy as they allow for
direct sampling of epileptogenic brain regions, assuming coverage is appropriate.
Localization of the seizure onset zone thus amounts to identifying the first contact
displaying epileptiform activity at seizure initiation. However, such activity often
appears nearly simultaneously on a number of contacts and visual identification
of relevant nodes of the EN is a challenging task. Thus, some strategies were pro-
posed for automatically labeling the most important contacts at seizure initiation.
By combining spectral and temporal information at seizure onset, Bartolomei
et al. introduced an empirical index that measures the propensity of each node to ini-
tiate seizures (Bartolomei et al., 2008). More specifically, they computed a ratio of
energy of high frequencies (beta and gamma) over lower frequency bands (delta,
theta, and alpha) at each time bin of the time-frequency decomposition of EEG sig-
nals. This ratio is then normalized and cumulated over time in what is called the
“energy ratio” function. When a seizure is recorded, this function contains a local
maximum at seizure onset, and the amplitude and latency of that maximum are used
to define the so-called epileptogenic index (EI), which measures the involvement in
seizure initiation.
The EI proved useful for the study on epileptogenic networks, especially in me-
sial TLE. Indeed, the number on nodes with high EI was higher for patients with
identified lesions than for patients with normal MRI, which was an important pre-
dictor of surgical outcome (Bartolomei et al., 2008). In addition, the EI captures
some subtleties in the clinical symptomatology of patients, since it can be used to
classify patients with clinical subtypes of mesial TLE (Bartolomei et al., 2010)
and discriminate patients with mesial MTLE from those suffering from other types
of TLE (Vaugier et al., 2009).
3.1.4 Neuroimaging brain networks

Networks can be schematized as a set of nodes connected to each other through spe-
cific links called edges. Nodes and edges are the workhorse of a large research com-
munity studying networks, ranging from air traffic, power plants to brain networks.
In neuroscience, network analyses answer two broad categories of questions: (1) how
does edge strength between a given node and a set of other nodes evolve with respect
to experimental paradigm and (2) how do the global features of networks evolve with
respect to experimental paradigm?
The main challenges here consist in defining relevant nodes and edges. Many
approaches were proposed for designing nodes encompassing random, data-driven
and atlas-based strategies. We note that atlas-based strategies provide regions of
interest (ROIs) that are more easily interpretable in terms of neurophysiology as
they allow for the understanding of neural systems in terms of associations of
broadly specialized functional units. Edges represent the strength of the connectiv-
ity between two nodes, and their interpretation depends on the imaging modality.
Briefly, we distinguish three types of connectivity: functional, effective, and struc-
tural. Functional and effective connectivity are measures of undirected and directed
statistical coupling among signals, respectively, and must be estimated using
multivariate time series (EEG, MEG, and fMRI). In turn, structural connectivity
is defined in terms of anatomical association between ROIs such as fiber tracts
density and is usually estimated through diffusion imaging. One can analyze the
values of edges linking a specific set of nodes, also called “seeds.” Those seed-
based analyses were carried out in a wide variety of epilepsy types, encompassing
“focal” and “generalized” types. In those analyses, variations in edge values are
assessed with respect to experimental paradigms, allowing the interpretation of
pathophysiology and clinical symptoms in terms of brain connectivity. A sample
of those studies is described in the following sections with respect to imaging
modalities.
The complete graph of connections between all available nodes is called a
“connectome” and analyses of such graph are thus termed “connectomics.” This
field of mathematics was originally framed into the so-called graph theory, which
recently raised spectacular interest in neuroscience in general, and epilepsy in par-
ticular. Graph theory provides a variety of metrics that describe interpretable fea-
tures of connectomes, such as the clustering coefficient, which indexes the
tendency of nodes to form “cliques” with dense internal connections, and the aver-
age path length, which measures the average number of relays while trying to go
from one node to another. Strikingly, many networks, including brain networks,
were found to have relatively high clustering coefficient and low average path
length. Those “small-world” networks share fast processing of information within
functional units through dense local connections and the ability to parallelize infor-
mation processing through sparse but efficient long-range connections (Watts and
Strogatz, 1998). In epilepsy, metrics such as “small-worldness,” efficiency, and
modularity shed new light on the large-scale neurophysiological mechanisms at play
during interictal and ictal states.
3.1.5 EEG/MEG connectivity

Definition of nodes and edges in EEG and MEG is usually done in the sources space
since each EEG electrode and MEG sensor records mixtures of neural signals from a
large portion of the brain, which implies that connectivity in the sensors spaces has
little interpretability with respect to underlying neural generators (but see Holmes
et al., 2010; Horstmann et al., 2010; van Mierlo et al., 2014 for interesting reports
on functional connectivity on the sensors). In the majority of studies, DSM is done
using thousands (10 k) of ECDs and connectivity analyses at such resolution is im-
practicable for two reasons: (1) the computational cost increases exponentially with
the number of sources and (2) the spatial resolution of ESI/MSI is much lower than
that of the head model, which implies that neighboring sources are heavily cross-
contaminated. The most common strategy to address these two issues is to perform
space reduction by pooling (Hillebrand et al., 2012; Tana et al., 2012) source signals
into ROIs, which are then taken as the nodes of the connectome. Connectivity among
ROIs can then be evaluated using various metrics, all of which have specific
strengths and weaknesses and they all display acceptable performance in the majority
of cases (Wendling et al., 2009).
When analyzing interictal discharges, few studies showed that functional connec-
tivity provides useful diagnostic information about the epileptic networks of patients
and even for surgical outcome prediction. Using ESI of interictal spikes and a time-
varying frequency-resolved effective connectivity metric, Coito et al. found dissoci-
ation in the pattern of connectivity between patients with left and right TLE (Coito
et al., 2015). Indeed, the latter exhibited increased contralateral connectivity, in line
with contralateral frontal functional deficits in right TLE. In addition, effective con-
nectivity on MEG data showed that surgical resection of the main driving nodes of
ENs could predict favorable outcome in 9/10 patients (Dai et al., 2012; Jin et al.,
2013). However, definite conclusions about outcome prediction are limited by the
fact that those studies did not include cases with negative surgical outcome. Simi-
larly, Malinowska et al. showed that MEG-identified network drivers showed good
concordance both with drivers identified from icEEG and with resected areas
(Malinowska et al., 2014).
Our group recently conducted a MEG study aimed at studying functional connec-
tivity networks at play during interictal insular spikes (Zerouali et al., accepted). We
computed the connectomes of insular spikes using MSI based on the maximum en-
tropy on the mean algorithm and phase synchronization measures and conducted
seed-based connectivity analyses. We showed that anterior and posterior parts of
the insula are characterized by markedly distinct connectivity networks, with the for-
mer connected mainly to anterior structures while the latter is mainly connected to
parietal and occipital structures (Fig. 6). In this study, we showed that MEG is able to
establish a robust signature of ICE based on functional connectivity measures, which
could open important avenues in the automatic detection of ICE.
Other studies analyzing seizures showed that transitions between brain states dis-
play deep modifications in connectivity. Using EEG and Granger causality, Coben
et al. showed hyperconnectivity at the transition between ictal and postictal states
FIG. 6
Functional connectivity of insular subregions during interictal spikes recorded with MEG and
quantified using the phase-locking value of narrow band-filtered signal (beta band,
12–30 Hz). Each insular subregion is represented in three panels (left, top, and right views),
with insular seeds appearing in white. Top row, anterior subregion; middle row, posterior
subregion; bottom row, inferior subregion. The color (gray shades in the print version) scale
encodes the strength of coupling insular seeds and the rest of the cortex, after statistical
thresholding.
(Coben and Mohammad-Rezazadeh, 2015). In addition, according to Elshoff et al.,

the pattern of connectivity at seizure initiation is entirely driven by a single node then
becomes circular around the middle of seizures (Elshoff et al., 2013). They also
showed that surgical resection of the main driving node at seizure initiation yielded
a positive outcome (8/8) while the opposite yielded a negative outcome (3/3) for pa-
tients. Interestingly, topological features were also shown to vary at those transitions.
Using frequency-resolved connectivity, Gupta et al. showed that transition from
preictal to ictal states display networks with increased “small-worldness,” which
suggests seizure initiation necessitates more ordered network structure (Gupta
et al., 2011).
3.1.6 fMRI connectivity

Early seed-based connectivity studies using fMRI were conducted during rest ses-
sions, where healthy subjects are instructed to avoid focusing on any particular
thoughts. Using such paradigm, Biswal et al. showed that bilateral motor cortices
are not silent but rather exhibit strong connectivity at rest, which suggests that these
regions continue sharing and processing information offline (Biswal et al., 1995,
1997). Later, other studies showed that large-scale brain connectivity at rest is rather
the norm than the exception, and a number of networks were found to be highly
consistent across subjects and experiments (see Fox and Raichle, 2007, for a review).
These resting-state networks are now well established as robust signatures of healthy
brain functioning.
Seed-based analysis was applied to study the functional connectivity of the hu-
man insula, revealing two main insular clusters, one anterior and one posterior. The
anterior portion of the insula (aI) was found to be connected to the anterior cingulate
cortex, the anterior and posterior parts of the middle cingulate cortex (MCC) while
the posterior portion of the insula (pI) was found to be connected only to the posterior
MCC (Taylor et al., 2009). In addition, the aI is functionally connected to the middle
and inferior frontal cortex while the pI is connected to the primary and secondary
somatosensory and the supplementary motor areas (Cauda et al., 2011). Some studies
refined this parcellation, showing that aI can be subdivided into ventral and dorsal aI,
each having specific patterns of functional connectivity with the cortex (Deen et al.,
2011). In addition, this tripartite parcellation is supported by the distinct involvement
of those three regions into specific cognitive tasks (Chang et al., 2013). However, to
our knowledge, no study has linked these specific connectivity networks to epileptic
activity in ICE. Such studies are needed to shed some new light on the pathophys-
iological mechanisms of that disease, as was done for other kinds of epilepsies.
3.1.7 Structural connectivity

The anatomical connections linking neural populations can be studied in vivo using
diffusion weighted imaging. This modality exploits information about the diffusivity
of molecules (mostly water) in the brain tissue. Mathematical models were proposed
to translate this information into 3D images of white fiber tracts. In general, these
models assume that water diffusivity in the brain is constrained by cellular structural
elements such that it has preferential directions, which can be estimated in the form
of fractional anisotropy (FA). For instance, water diffuses much more freely along
than across the axon, thus a voxel crossed by an axonal bundle would have a high FA.
Importantly, by tracking the FA along consecutive voxels, thereby performing trac-
tography, it is possible to reconstruct the major white fiber tracts of the brain (Jbabdi
and Johansen-Berg, 2011; Le Bihan, 2003; Mori et al., 2002).
To our knowledge, only three studies used tractography to investigate the struc-
tural connectivity of the insula. They report comparable connectivity profiles show-
ing that the anterior insular cortex has connections mostly with frontal and temporal
(inferior and superior gyri, amygdala) structures. The middle insular cortex has con-
nections with frontal (superior, inferior, and precentral), parietal (postcentral and
supramarginal), and temporal (inferior and superior) gyri. Finally, the posterior in-
sular cortex has connections with frontal (superior, inferior, and precentral), parietal
(postcentral), and temporal (inferior and superior) gyri and with the putamen
(Cerliani et al., 2012; Cloutman et al., 2012; Jakab et al., 2012). These results
are in accordance with tract-tracing studies but some connections in primates
were not found in humans. Using state-of-the-art tractography, our group further
investigated the structural connectivity of the human insula and found many previ-
ously missed connections, such as those with the cingulate, parahippocampal,
supramarginal, angular, and lingual gyri as well as the precuneus, cuneus, and occip-
ital cortex (Ghaziri et al., 2015).
Tractography can also be used to study white matter insults in relation to epilepsy
(for a review, see Anastasopoulos et al., 2014; Ciccarelli et al., 2008; Winston, 2015).
Studies generally report a FA decrease and a mean diffusivity increase in pathways
near the epileptogenic temporal lobe (eg, optic radiations, uncinate and arcuate fas-
ciculi, cingulum, fornix, and external capsule), which reflects reduced axonal density
in TLE. White matter insults in TLE are mostly ipsilateral to the SOZ and tracts
closely connected to the affected temporal lobe are the most disturbed. Furthermore,
fiber tracts remote from the temporal lobe are also affected, which supports the view
of epilepsy as a brain network disease (Concha et al., 2012; Gross, 2011; Otte et al.,
2012; Rodrı́guez-Cruces and Concha, 2015). To follow-up on our study on healthy
controls, we performed tractography on patients with ICE. Using nonparametric sta-
tistical tests, we assessed the differences between each patient and controls in fiber
tract density linking the insula to the remaining cortex. Although preliminary, our
results suggest that anterior, posterior, and inferior ICE differentially affects the
white fiber tracts, which could open new perspectives in the diagnosis of this kind
of epilepsy.
3.1.8 icEEG connectivity

The main specificity of icEEG as compared to EEG/MEG connectivity is the sparse
sampling of neural generators, and coverage extent is determined by balancing the
amount of diagnostic information and health risks for patients. For that reason, it is
usually agreed that icEEG connectivity only allows partial assessment of the brain
connectome. However, judicious choice of epileptic cases in conjunction with the
optimized placement of electrodes allows for characterizing the most relevant as-
pects of ENs. Indeed, most of the studies we describe below are conducted on pa-
tients with focal seizure onsets and limited propagation, such that the EN can
mostly be sampled with intracranial electrodes.
Since the introduction of the network concept of epilepsy in the early 2000s, a
large research effort was devoted to characterizing neural synchronization related
to the epilepsy. The unequivocal findings of such research are that (1) from a static
point of view, the epileptic condition is characterized by deep impacts on large-scale
neural synchronization and (2) the most spectacular changes in neural synchroniza-
tion are related to brain dynamics, ie, occur at transitions between consecutive brain
states before, during, and after seizures. In the following, we discuss the literature
relative to those two findings separately.
3.1.8.1 Static network properties

In order to study the impact of the epileptic condition on brain networks, the most
common paradigm consists in using artifact-free rest EEG data. Using such data, two
independent studies showed that patterns of neural synchronization characterize dis-
tinct groups of epileptic patients. Ortega et al. analyzed ECoG data from 29 patients
with TLE and computed three synchronization measures in a short time frame sliding
over continuous recordings (Ortega et al., 2008). They showed that synchronized
ECoG contacts can either spread over the whole lateral temporal lobe or cluster
tightly in specific subregions. Importantly, they found that the predictive value with
respect to seizure-freedom after surgery (Engel Ia) was very high and very low for
tight and diffuse synchronization clusters, respectively. This suggests that synchro-
nization patterns can be used to identify regions participating in seizures.
In addition to spatial patterns, synchronization strength was also shown to predict
surgical outcome. In a series of 29 patients with TLE, Antony et al. assessed synchro-
nization strengths among EEG signals recorded with intracerebral electrodes at rest.
They showed that patients with low connectivity strength had better surgical out-
come than patients with high connectivity strength, and that the linear classifier
was able to accurately classify those two groups of patients based on the average
and standard deviation of global synchronization (Antony et al., 2013). The idea that
decreased levels of synchronization might be characteristic of the epileptic condition
received further support when Warren et al. (2010) compared synchrony at rest be-
tween epileptic patients and controls with intracranial electrodes implanted for treat-
ing intractable facial pain. Controlling for intercontact distance, synchrony levels
between patients and controls were either decreased or increased depending on
the frequency band analyzed. However, finer analysis revealed that synchrony be-
tween the SOZ and other brain regions is significantly weaker than in controls, while
synchrony either within SOZ or outside SOZ was unchanged (Warren et al., 2010).
Further insights into the role of synchrony in epilepsy were provided by studies of
seizure dynamics.
3.1.8.2 Network dynamics: Synchrony

In order to study temporal evolution of synchrony levels, Wendling et al. recorded
seizures with icEEG in 10 patients with focal epilepsy. Comparing global synchrony
levels (as measured with a linear correlation coefficient), they showed that seizure
initiation displays large decreases in synchrony as compared to preictal and postictal
states (Wendling, 2003). In another study on patients with medial TLE, Mormann
et al. showed that synchrony levels between bilateral hippocampi are markedly de-
creased before seizure onset and return gradually to baseline levels as seizure unfolds
(Mormann et al., 2003). Interestingly, in 8 out of 10 patients, they were able to ac-
curately predict seizures by detecting preseizure state based on reported lower syn-
chronization values.
In addition, some authors tracked synchrony levels in epileptic networks along
seizures. Using icEEG recordings from 11 patients with focal epilepsy, Kramer
et al. performed temporal normalization for aligning seizures from different patients
into 10 consecutive windows (each covering 10% of the seizure). In contradiction
to the two previous studies, they found a steep increase in synchrony levels in the
first and last windows during seizures (Kramer et al., 2010). However, they also
found that seizures were characterized by networks with constant nodal degree
and small-world topology, while the transition from ictal to postictal state was char-
acterized by highly increased nodal degree and randomness. Further refining this
strategy, Burns et al. used a data-driven approach to represent network dynamics

with a finite set of representative networks. Their main result is that for some pa-
tients, there exists two states during which the SOZ is specifically disconnected (iso-
lated focus, IF, state) and overconnected (connected focus—CF), respectively. When
present, the IF state occurred at seizure initiation and lasted for about the first half of
seizures. Importantly, patients for whom IF state could be detected had significantly
better surgical outcome than patients for whom IF state was not detected (Burns
et al., 2014). This important study shows that the epileptic zones that specifically
detach from the EN at seizure initiation are good candidates for surgical resection.
Further refinement of the synchrony analyses reviewed here is possible by consid-
ering the directionality of connections through the assessment of effective
connectivity.
3.1.8.3 Network dynamics: Directionality

Formal distinction between contributions of outgoing and incoming connections in
EN analysis was provided by Varotto et al. who compared the connectivity of intra-
cranial electrode contacts in 10 patients with lesional epilepsy. They divided contacts
into three groups: within the lesion (LES), involved in the seizure but outside the
lesion (INV), and not involved in seizures (NINV) and showed that outgoing con-
nections of LES and INV contacts increase significantly at seizure onset. Interest-
ingly, while outgoing LES connections were directed toward the INV group,
outgoing connections of the INV group were diffuse. In contrast, incoming connec-
tions did not show significant differences between the three groups (Varotto et al.,
2012). In the same vein, it was shown that blind identification of the electrode contact
with the highest number of outgoing connections at seizure initiation localized the
surgically defined SOZ in two patient series (8/8, van Mierlo et al., 2013; 11/11,
Wilke et al., 2010). Finally, there was also a good correlation between the percentage
of connections exiting nodes localized within SOZ and percentage of seizure reduc-
tion after surgery (Wilke et al., 2010).
3.1.8.4 Network dynamics: The case of ICE

Since the early 2000s, our group started sampling insular regions with intracranial
electrodes systematically when patients were suspected of either frontal, temporal,
or parietal lobe epilepsy with inconsistent clinical semiology. We thus constituted
a large database of ICE cases including icEEG recordings and recently started ana-
lyzing network dynamics and topology. We found that most significant changes in
effective connectivity were found in the gamma band (Fig. 7), more specifically at
seizure initiation, electrographic shift, and termination (Fig. 8). In addition, insular
contacts were characterized by an increase in outgoing connections at seizure initi-
ation, followed by disconnection. These results are in agreement with those reported
by Burns et al. (2014), which suggests that an IF state is present in patients with ICE.
This interpretation is supported by the fact that most of our ICE cases were seizure
free after insulectomy.
FIG. 7
Effective connectivity at seizure onset computed from intracranial EEG data on three patients with insular cortex epilepsy. Effective connectivity is
computed using the directed phase-lag index (Stam and van Straaten, 2012) in narrow band-filtered signals in the following frequencies: theta
(4–7 Hz), alpha (8–12 Hz), beta (12–30 Hz), and gamma (30–90 Hz). Top row, raw connectivity matrices; bottom row, statistical threshold
applied using the false-discovery rate technique. The null hypothesis was modeled using baseline data segments recorded 2 min before the
beginning of seizures.
FIG. 8
Network dynamics of insular cortex epilepsy. Thresholded connectivity matrices in the
gamma band are displayed at transitions between brain states along seizures. At seizure
initiation, the seizure onset zone (ie, the insula—electrode U1, U3) is the main driving node of
the epileptogenic network. At the transition from low-voltage fast activity to high-amplitude
slow oscillations, the insula detaches from the network and remains detached until seizure
termination, which is marked by a dense and unstructured connectivity network.
4 CONCLUSION
Insular cortex epilepsy is a challenging disease that can easily be mistaken for other
forms of epilepsy for several reasons. First, clinical semiology is often confusing
since the insula may generate a variety of symptoms classically associated with other
types of focal epilepsy and because insular seizures can be asymptomatic until par-
oxysmal activity propagates to secondary brain structures. Second, standard clinical
neuroimaging tests often fail to detect this type of epilepsy; no single noninvasive
test is accurate enough to provide an efficient biomarker of insular cortex seizures
or spikes. However, the most informative noninvasive tests are T1-contrast MRI im-
aging and sECD modeling of insular epileptic spikes, and consensus between MRI/
MEG and any other test can reach the clinical threshold for proceeding with insulect-
omy without recording icEEG.
Our current understanding of the functional networks involved in insular epilepsy
is still mostly descriptive but ongoing efforts from our group and others have the po-
tential to provide clinically useful information. Using MEG, we could establish func-
tional connectivity-based signatures of two subtypes of ICE and we are currently
investigating the potential of the signatures as biomarkers of ICE. In addition, inves-
tigating the causal relationships during seizures with icEEG, we found that the insula
is necessary for initiating seizures but not for its maintenance. We are currently pur-
suing these investigations to relate the causal role of the insula during seizures with
References 23
surgical outcomes following insulectomy, with the aim to fine-tune the surgical ap-
proach and optimize its success.
REFERENCES
Aghakhani, Y., 2004. FMRI activation during spike and wave discharges in idiopathic gener-
alized epilepsy. Brain 127, 1127–1144. http://dx.doi.org/10.1093/brain/awh136.
Aghakhani, Y., Rosati, A., Dubeau, F., Olivier, A., Andermann, F., 2004. Patients with tem-
poroparietal ictal symptoms and inferomesial EEG do not benefit from anterior temporal
resection. Epilepsia 45, 230–236.
Aguirre, G.K., Zarahn, E., D’esposito, M., 1998. The variability of human, BOLD hemody-
namic responses. NeuroImage 8, 360–369. http://dx.doi.org/10.1006/nimg.1998.0369.
Ahlfors, S.P., Han, J., Belliveau, J.W., H€am€al€ainen, M.S., 2010. Sensitivity of MEG and EEG
to source orientation. Brain Topogr. 23, 227–232. http://dx.doi.org/10.1007/s10548-010-
0154-x.
Allen, P.J., Josephs, O., Turner, R., 2000. A method for removing imaging artifact from con-
tinuous EEG recorded during functional MRI. NeuroImage 12, 230–239. http://dx.doi.org/
10.1006/nimg.2000.0599.
Amblard, C., Lapalme, E., Lina, J.-M., 2004. Biomagnetic source detection by maximum en-
tropy and graphical models. IEEE Trans. Biomed. Eng. 51, 427–442. http://dx.doi.org/
10.1109/TBME.2003.820999.
An, D., Fahoum, F., Hall, J., Olivier, A., Gotman, J., Dubeau, F., 2013. Electroencephalogra-
phy/functional magnetic resonance imaging responses help predict surgical outcome in
focal epilepsy. Epilepsia 54, 2184–2194. http://dx.doi.org/10.1111/epi.12434.
Anastasopoulos, C., Reisert, M., Kiselev, V.G., Nguyen-Thanh, T., Schulze-Bonhage, A.,
Zentner, J., Mader, I., 2014. Local and global fiber tractography in patients with epilepsy.
Am. J. Neuroradiol. 35, 291–296. http://dx.doi.org/10.3174/ajnr.A3752.
Antony, A.R., Alexopoulos, A.V., GonzÃ¡lez-MartÃ-nez, J.A., Mosher, J.C., Jehi, L.,
Burgess, R.C., So, N.K., GalÃ¡n, R.F., 2013. Functional connectivity estimated from
intracranial EEG predicts surgical outcome in intractable temporal lobe epilepsy. PLoS
One 8, e77916. http://dx.doi.org/10.1371/journal.pone.0077916.
Augustine, J.R., 1996. Circuitry and functional aspects of the insular lobe in primates includ-
ing humans. Brain Res. Brain Res. Rev. 22, 229–244.
Avanzini, G., Franceschetti, S., 2003. Cellular biology of epileptogenesis. Lancet Neurol.
2, 33–42.
Avesani, M., Giacopuzzi, S., Bongiovanni, L.G., Borelli, P., Cerini, R., Pozzi Mucelli, R.,
Fiaschi, A., 2014. EEG-fMRI evaluation of patients with mesial temporal lobe sclerosis.
Neuroradiol. J. 27, 45. http://dx.doi.org/10.15274/NRJ-2014-10005.
€ Vorwerk, J., D€umpelmann, M., K€upper, P., Kugel, H., Heers, M., Wellmer, J.,
Aydin, U.,
Kellinghaus, C., Haueisen, J., Rampp, S., Stefan, H., Wolters, C.H., 2015. Combined
EEG/MEG can outperform single modality EEG or MEG source reconstruction in Presur-
gical epilepsy diagnosis. PLoS One 10, e0118753. http://dx.doi.org/10.1371/journal.
pone.0118753.
Barkley, G.L., Baumgartner, C., 2003. MEG and EEG in epilepsy. J. Clin. Neurophysiol.
20, 163–178.
Bartolomei, F., Chauvel, P., Wendling, F., 2008. Epileptogenicity of brain structures in human
temporal lobe epilepsy: a quantified study from intracerebral EEG. Brain 131, 1818–1830.
http://dx.doi.org/10.1093/brain/awn111.
Bartolomei, F., Cosandier-Rimele, D., McGonigal, A., Aubert, S., Regis, J., Gavaret, M.,
Wendling, F., Chauvel, P., 2010. From mesial temporal lobe to temporoperisylvian
seizures: a quantified study of temporal lobe seizure networks: epileptogenic brain net-
works in TLE. Epilepsia 51, 2147–2158. http://dx.doi.org/10.1111/j.1528-
1167.2010.02690.x.
Binder, D.K., Podlogar, M., Clusmann, H., Bien, C., Urbach, H., Schramm, J., Kral, T., 2009.
Surgical treatment of parietal lobe epilepsy: clinical article. J. Neurosurg. 110, 1170–1178.
http://dx.doi.org/10.3171/2008.2.17665.
Biswal, B., Yetkin, F.Z., Haughton, V.M., Hyde, J.S., 1995. Functional connectivity in the
motor cortex of resting human brain using echo-planar MRI. Magn. Reson. Med.
34, 537–541.
Biswal, B.B., Van Kylen, J., Hyde, J.S., 1997. Simultaneous assessment of flow and BOLD
signals in resting-state functional connectivity maps. NMR Biomed. 10, 165–170.
Boon, P., D’Have, M., Vanrumste, B., Van Hoey, G., Vonck, K., Van Walleghem, P.,
Caemaert, J., Achten, E., De Reuck, J., 2002. Ictal source localization in presurgical pa-
tients with refractory epilepsy. J. Clin. Neurophysiol. 19, 461–468.
Bouilleret, V., Dupont, S., Spelle, L., Baulac, M., Samson, Y., Semah, F., 2002. Insular cortex
involvement in mesiotemporal lobe epilepsy: a positron emission tomography study. Ann.
Neurol. 51, 202–208. http://dx.doi.org/10.1002/ana.10087.
Bragin, A., Engel, J., Wilson, C.L., Fried, I., Buzsáki, G., 1999. High-frequency oscillations in
human brain. Hippocampus 9, 137–142. http://dx.doi.org/10.1002/(SICI)1098-1063
(1999)9:2<137::AID-HIPO5>3.0.CO;2-0.
Breakspear, M., Roberts, J.A., Terry, J.R., Rodrigues, S., Mahant, N., Robinson, P.A., 2006.
A unifying explanation of primary generalized seizures through nonlinear brain modeling
and bifurcation analysis. Cereb. Cortex 1991 (16), 1296–1313. http://dx.doi.org/10.1093/
cercor/bhj072.
Brodbeck, V., Spinelli, L., Lascano, A.M., Wissmeier, M., Vargas, M.-I., Vulliemoz, S.,
Pollo, C., Schaller, K., Michel, C.M., Seeck, M., 2011. Electroencephalographic source
imaging: a prospective study of 152 operated epileptic patients. Brain 134, 2887–2897.
http://dx.doi.org/10.1093/brain/awr243.
Bronen, R.A., Fulbright, R.K., Spencer, D.D., Spencer, S.S., Kim, J.H., Lange, R.C.,
Sutilla, C., 1996. Refractory epilepsy: comparison of MR imaging, CT, and histopatho-
logic findings in 117 patients. Radiology 201, 97–105. http://dx.doi.org/10.1148/
radiology.201.1.8816528.
Brooks, B.S., King, D.W., el Gammal, T., Meador, K., Yaghmai, F., Gay, J.N., Smith, J.R.,
Flanigin, H.F., 1990. MR imaging in patients with intractable complex partial epileptic
seizures. AJNR Am. J. Neuroradiol. 11, 93–99.
Burns, S.P., Santaniello, S., Yaffe, R.B., Jouny, C.C., Crone, N.E., Bergey, G.K.,
Anderson, W.S., Sarma, S.V., 2014. Network dynamics of the brain and influence of
the epileptic seizure onset zone. Proc. Natl. Acad. Sci. 111, E5321–E5330. http://dx.
doi.org/10.1073/pnas.1401752111.
Buxton, R.B., Wong, E.C., Frank, L.R., 1998. Dynamics of blood flow and
oxygenation changes during brain activation: the balloon model. Magn. Reson. Med.
39, 855–864.
Cascino, G.D., Jack, C.R., Hirschorn, K.A., Sharbrough, F.W., 1992. Identification of the
epileptic focus: magnetic resonance imaging. Epilepsy Res. Suppl. 5, 95–100.
Cauda, F., D’Agata, F., Sacco, K., Duca, S., Geminiani, G., Vercelli, A., 2011. Functional
connectivity of the insula in the resting brain. NeuroImage 55, 8–23. http://dx.doi.org/
10.1016/j.neuroimage.2010.11.049.
References 25
Cerliani, L., Thomas, R.M., Jbabdi, S., Siero, J.C., Nanetti, L., Crippa, A., Gazzola, V.,
D’Arceuil, H., Keysers, C., 2012. Probabilistic tractography recovers a rostrocaudal trajectory
of connectivity variability in the human insular cortex. Hum. Brain Mapp. 33, 2005–2034.
Chang, L.J., Yarkoni, T., Khaw, M.W., Sanfey, A.G., 2013. Decoding the role of the insula in
human cognition: functional parcellation and large-scale reverse inference. Cereb. Cortex
23, 739–749. http://dx.doi.org/10.1093/cercor/bhs065.
Chowdhury, R.A., Lina, J.M., Kobayashi, E., Grova, C., 2013. MEG source localization of
spatially extended generators of epileptic activity: comparing entropic and hierarchical
Bayesian approaches. PLoS One 8, e55969. http://dx.doi.org/10.1371/journal.
pone.0055969.
Chowdhury, R.A., Zerouali, Y., Hedrich, T., Heers, M., Kobayashi, E., Lina, J.-M., Grova, C.,
2015. MEG–EEG information fusion and electromagnetic source imaging: from theory to
clinical application in epilepsy. Brain Topogr. 28, 785–812. http://dx.doi.org/10.1007/
s10548-015-0437-3.
Ciccarelli, O., Catani, M., Johansen-Berg, H., Clark, C., Thompson, A., 2008. Diffusion-based
tractography in neurological disorders: concepts, applications, and future developments.
Lancet Neurol. 7, 715–727. http://dx.doi.org/10.1016/S1474-4422(08)70163-7.
Cloutman, L.L., Binney, R.J., Drakesmith, M., Parker, G.J., Lambon Ralph, M.A., 2012. The
variation of function across the human insula mirrors its patterns of structural connectivity:
evidence from in vivo probabilistic tractography. Neuroimage 59, 3514–3521.
Coben, R., Mohammad-Rezazadeh, I., 2015. Neural connectivity in epilepsy as measured
by granger causality. Front. Hum. Neurosci. 9, http://dx.doi.org/10.3389/fnhum.2015.
00194.
Coito, A., Plomp, G., Genetti, M., Abela, E., Wiest, R., Seeck, M., Michel, C.M.,
Vulliemoz, S., 2015. Dynamic directed interictal connectivity in left and right temporal
lobe epilepsy. Epilepsia 56, 207–217. http://dx.doi.org/10.1111/epi.12904.
Concha, L., Kim, H., Bernasconi, A., Bernhardt, B.C., Bernasconi, N., 2012. Spatial patterns
of water diffusion along white matter tracts in temporal lobe epilepsy. Neurology
79, 455–462. http://dx.doi.org/10.1212/WNL.0b013e31826170b6.
Cukiert, A., Forster, C., ANDRIOLI, M.S., Frayman, L., 1998. Insular epilepsy: similarities to
temporal lobe epilepsy case report. Arq. Neuropsiquiatr. 56, 126–128.
D’Esposito, M., Zarahn, E., Aguirre, G.K., Rypma, B., 1999. The effect of normal aging on the
coupling of neural activity to the bold hemodynamic response. NeuroImage 10, 6–14.
http://dx.doi.org/10.1006/nimg.1999.0444.
Da Silva, F.L., Blanes, W., Kalitzin, S.N., Parra, J., Suffczynski, P., Velis, D.N., 2003. Epi-
lepsies as dynamical diseases of brain systems: basic models of the transition between nor-
mal and epileptic activity. Epilepsia 44, 72–83.
Dai, Y., Zhang, W., Dickens, D.L., He, B., 2012. Source connectivity analysis from MEG and
its application to epilepsy source localization. Brain Topogr. 25, 157–166. http://dx.doi.
org/10.1007/s10548-011-0211-0.
Debets, R.M., Sadzot, B., Van Isselt, J.W., Brekelmans, G.J., Meiners, L.C., Van Huffelen,
A.O., Franck, G., Van Veelen, C.W., 1997. Is 11C-flumazenil PET superior to 18FDG
PET and 123I-iomazenil SPECT in presurgical evaluation of temporal lobe epilepsy?
J. Neurol. Neurosurg. Psychiatry 62, 141–150.
Deen, B., Pitskel, N.B., Pelphrey, K.A., 2011. Three systems of insular functional connectivity
identified with cluster analysis. Cereb. Cortex 21, 1498–1506. http://dx.doi.org/10.1093/
cercor/bhq186.
Desai, A., Bekelis, K., Thadani, V.M., Roberts, D.W., Jobst, B.C., Duhaime, A.-C., Gilbert, K.,
Darcey, T.M., Studholme, C., Siegel, A., 2013. Interictal PET and ictal subtraction
SPECT: sensitivity in the detection of seizure foci in patients with medically intractable
epilepsy. Interictal PET and Ictal Subtraction SPECT, Epilepsia 54, 341–350. http://dx.
doi.org/10.1111/j.1528-1167.2012.03686.x.
Didelot, A., Ryvlin, P., Lothe, A., Merlet, I., Hammers, A., Mauguiere, F., 2008. PET imaging
of brain 5-HT1A receptors in the preoperative evaluation of temporal lobe epilepsy. Brain
131, 2751–2764. http://dx.doi.org/10.1093/brain/awn220.
Dobesberger, J., Ortler, M., Unterberger, I., Walser, G., Falkenstetter, T., Bodner, T.,
Benke, T., Bale, R., Fiegele, T., Donnemiller, E., Gotwald, T., Trinka, E., 2008. Successful
surgical treatment of insular epilepsy with nocturnal hypermotor seizures. Epilepsia
49, 159–162. http://dx.doi.org/10.1111/j.1528-1167.2007.01426.x.
Duffau, H., Capelle, L., Lopes, M., Bitar, A., Sichez, J.-P., van Effenterre, R., 2002. Medically
intractable epilepsy from insular Low-grade gliomas: improvement after an extended
lesionectomy. Acta Neurochir. (Wien) 144, 563–573. http://dx.doi.org/10.1007/s00701-
002-0941-6.
Ebersole, J.S., 1991. EEG dipole modeling in complex partial epilepsy. Brain Topogr.
4, 113–123.
Ebersole, J.S., Ebersole, S.M., 2010. Combining MEG and EEG source modeling in epilepsy
evaluations. J. Clin. Neurophysiol. 27, 360–371.
Elshoff, L., Muthuraman, M., Anwar, A.R., Deuschl, G., Stephani, U., Raethjen, J.,
Siniatchkin, M., 2013. Dynamic imaging of coherent sources reveals different network
connectivity underlying the generation and perpetuation of epileptic seizures. PLoS
One 8, e78422. http://dx.doi.org/10.1371/journal.pone.0078422.
Fischer, M.J.M., Scheler, G., Stefan, H., 2005. Utilization of magnetoencephalography results
to obtain favourable outcomes in epilepsy surgery. Brain J. Neurol. 128, 153–157. http://
dx.doi.org/10.1093/brain/awh333.
Fox, M.D., Raichle, M.E., 2007. Spontaneous fluctuations in brain activity observed with
functional magnetic resonance imaging. Nat. Rev. Neurosci. 8, 700–711. http://dx.doi.
org/10.1038/nrn2201.
Friston, K., 1995. Analysis of fMRI time-series revisited. NeuroImage 2, 45–53. http://dx.doi.
org/10.1006/nimg.1995.1007.
Friston, K.J., Fletcher, P., Josephs, O., Holmes, A., Rugg, M.D., Turner, R., 1998. Event-
related fMRI: characterizing differential responses. NeuroImage 7, 30–40. http://dx.doi.
org/10.1006/nimg.1997.0306.
Fujiwara, H., Greiner, H.M., Hemasilpin, N., Lee, K.H., Holland-Bouley, K., Arthur, T.,
Morita, D., Jain, S.V., Mangano, F.T., Degrauw, T., Rose, D.F., 2012. Ictal MEG onset
source localization compared to intracranial EEG and outcome: improved epilepsy presur-
gical evaluation in pediatrics. Epilepsy Res. 99, 214–224. http://dx.doi.org/10.1016/j.
eplepsyres.2011.11.007.
Ghaziri, J., Tucholka, A., Girard, G., Houde, J.C., Boucher, O., Gilbert, G., Descoteaux, M.,
Lippe, S., Rainville, P., Nguyen, D.K., 2015. The corticocortical structural connectivity of
the human insula. Cereb. Cortex. [Epub ahead of print].
Gonçalves, S.I., Pouwels, P.J.W., Kuijer, J.P.A., Heethaar, R.M., de Munck, J.C., 2007. Ar-
tifact removal in co-registered EEG/fMRI by selective average subtraction. Clin. Neuro-
physiol. 118, 2437–2450. http://dx.doi.org/10.1016/j.clinph.2007.08.017.
Grattan-Smith, J.D., Harvey, A.S., Desmond, P.M., Chow, C.W., 1993. Hippocampal sclerosis
in children with intractable temporal lobe epilepsy: detection with MR imaging. AJR Am.
J. Roentgenol. 161, 1045–1048. http://dx.doi.org/10.2214/ajr.161.5.8273606.
Gross, D.W., 2011. Diffusion tensor imaging in temporal lobe epilepsy: DTI in temporal lobe
epilepsy. Epilepsia 52, 32–34. http://dx.doi.org/10.1111/j.1528-1167.2011.03149.x.
References 27
Grova, C., Daunizeau, J., Lina, J.-M., Benar, C.G., Benali, H., Gotman, J., 2006. Evaluation of
EEG localization methods using realistic simulations of interictal spikes. NeuroImage
29, 734–753. http://dx.doi.org/10.1016/j.neuroimage.2005.08.053.
Guillaume, M., Mazars, G., 1949. Cinq cas de foyers epileptogènes insulaires operes. Soci et
e
Française de Neurologie, pp. 766–769.
Gupta, D., Ossenblok, P., van Luijtelaar, G., 2011. Space–time network connectivity and cor-
tical activations preceding spike wave discharges in human absence epilepsy: a MEG
study. Med. Biol. Eng. Comput. 49, 555–565. http://dx.doi.org/10.1007/s11517-011-
0778-3.
Halász, P., 2010. The concept of epileptic networks. Part 1. Ideggyógy. Szle 63, 293–303.
Handwerker, D.A., Ollinger, J.M., D’Esposito, M., 2004. Variation of BOLD hemodynamic
responses across subjects and brain regions and their effects on statistical analyses.
NeuroImage 21, 1639–1651. http://dx.doi.org/10.1016/j.neuroimage.2003.11.029.
Heers, M., Rampp, S., Stefan, H., Urbach, H., Elger, C.E., von Lehe, M., Wellmer, J., 2012.
MEG-based identification of the epileptogenic zone in occult peri-insular epilepsy.
Seizure 21, 128–133. http://dx.doi.org/10.1016/j.seizure.2011.10.005.
Hillebrand, A., Barnes, G.R., Bosboom, J.L., Berendse, H.W., Stam, C.J., 2012. Frequency-
dependent functional connectivity within resting-state networks: an atlas-based MEG
beamformer solution. NeuroImage 59, 3909–3921. http://dx.doi.org/10.1016/j.
neuroimage.2011.11.005.
Holmes, M.D., Quiring, J., Tucker, D.M., 2010. Evidence that juvenile myoclonic epilepsy is a
disorder of frontotemporal corticothalamic networks. NeuroImage 49, 80–93. http://dx.
doi.org/10.1016/j.neuroimage.2009.08.004.
Horstmann, M.-T., Bialonski, S., Noennig, N., Mai, H., Prusseit, J., Wellmer, J., Hinrichs, H.,
Lehnertz, K., 2010. State dependent properties of epileptic brain networks: comparative
graph–theoretical analyses of simultaneously recorded EEG and MEG. Clin. Neurophy-
siol. 121, 172–185. http://dx.doi.org/10.1016/j.clinph.2009.10.013.
Hur, J.A., Kang, J.W., Kang, H.-C., Kim, H.D., Kim, J.T., Lee, J.S., 2013. The significance of
insular hypometabolism in temporal lobe epilepsy in children. J. Epilepsy Res. 3, 54.
Isnard, J., Guenot, M., Ostrowsky, K., Sindou, M., Mauguière, F., 2000. The role of the insular
cortex in temporal lobe epilepsy. Ann. Neurol. 48, 614–623. http://dx.doi.org/
10.1002/1531-8249(200010)48:4<614::AID-ANA8>3.0.CO;2-S.
Isnard, J., Guenot, M., Sindou, M., Mauguière, F., 2004. Clinical manifestations of insular lobe
seizures: a stereo-electroencephalographic study. Epilepsia 45, 1079–1090. http://dx.doi.
org/10.1111/j.0013-9580.2004.68903.x.
Iwasaki, M., Pestana, E., Burgess, R.C., L€uders, H.O., Shamoto, H., Nakasato, N., 2005. De-
tection of epileptiform activity by human interpreters: blinded comparison between elec-
troencephalography and magnetoencephalography. Epilepsia 46, 59–68. http://dx.doi.org/
10.1111/j.0013-9580.2005.21104.x.
Jacobs, J., Hawco, C., Kobayashi, E., Boor, R., LeVan, P., Stephani, U., Siniatchkin, M.,
Gotman, J., 2008. Variability of the hemodynamic response as a function of age and fre-
quency of epileptic discharge in children with epilepsy. NeuroImage 40, 601–614. http://
dx.doi.org/10.1016/j.neuroimage.2007.11.056.
Jakab, A., Molnár, P.P., Bogner, P., Beres, M., Berenyi, E.L., 2012. Connectivity-based par-
cellation reveals interhemispheric differences in the insula. Brain Topogr. 25, 264–271.
Jbabdi, S., Johansen-Berg, H., 2011. Tractography: where do we go from here? Brain Connect.
1, 169–183. http://dx.doi.org/10.1089/brain.2011.0033.
Jeha, L.E., Najm, I.M., Bingaman, W.E., Khandwala, F., Widdess-Walsh, P., Morris, H.H.,
Dinner, D.S., Nair, D., Foldvary-Schaeffer, N., Prayson, R.A., Comair, Y., O’Brien, R.,
Bulacio, J., Gupta, A., L€uders, H.O., 2006. Predictors of outcome after temporal lobec-
tomy for the treatment of intractable epilepsy. Neurology 66, 1938–1940. http://dx.doi.
org/10.1212/01.wnl.0000219810.71010.9b.
Jeha, L.E., Najm, I., Bingaman, W., Dinner, D., Widdess-Walsh, P., L€ uders, H., 2007. Surgical
outcome and prognostic factors of frontal lobe epilepsy surgery. Brain J. Neurol.
130, 574–584. http://dx.doi.org/10.1093/brain/awl364.
Jin, S.-H., Jeong, W., Chung, C.K., 2013. Information source in multiple MEG spike clusters
can be identified by effective connectivity in focal cortical dysplasia. Epilepsy Res.
105, 118–124. http://dx.doi.org/10.1016/j.eplepsyres.2013.01.011.
Joo, E.Y., 2005. Postoperative alteration of cerebral glucose metabolism in mesial temporal
lobe epilepsy. Brain 128, 1802–1810. http://dx.doi.org/10.1093/brain/awh534.
Joo, E.Y., Hong, S.B., Lee, E.K., Tae, W.S., Kim, J.H., Seo, D.W., Hong, S.C., Kim, S.,
Kim, M.-H., 2004. Regional cerebral hyperperfusion with ictal dystonic posturing:
ictal-interictal SPECT subtraction. Epilepsia 45, 686–689. http://dx.doi.org/10.1111/
j.0013-9580.2004.35003.x.
Josephs, O., Henson, R.N.A., 1999. Event-related functional magnetic resonance imaging:
modelling, inference and optimization. Philos. Trans. R. Soc. B Biol. Sci.
354, 1215–1228. http://dx.doi.org/10.1098/rstb.1999.0475.
Juergens, E., Guettler, A., Eckhorn, R., 1999. Visual stimulation elicits locked and induced
gamma oscillations in monkey intracortical- and EEG-potentials, but not in human
EEG. Exp. Brain Res. 129, 247–259.
Kaiboriboon, K., L€uders, H.O., Hamaneh, M., Turnbull, J., Lhatoo, S.D., 2012. EEG source
imaging in epilepsy—practicalities and pitfalls. Nat. Rev. Neurol. 8, 498–507. http://dx.
doi.org/10.1038/nrneurol.2012.150.
Kaido, T., Otsuki, T., Nakama, H., Kaneko, Y., 2006. Hypermotor seizure arising from insular
cortex. Epilepsia 47, 1587–1588. http://dx.doi.org/10.1111/j.1528-1167.2006.00843_4.x.
Kim, D.W., Lee, S.K., Yun, C.-H., Kim, K.-K., Lee, D.S., Chung, C.-K., Chang, K.-H., 2004a.
Parietal lobe epilepsy: the semiology, yield of diagnostic workup, and surgical outcome.
Epilepsia 45, 641–649. http://dx.doi.org/10.1111/j.0013-9580.2004.33703.x.
Kim, K.H., Yoon, H.W., Park, H.W., 2004b. Improved ballistocardiac artifact removal from
the electroencephalogram recorded in fMRI. J. Neurosci. Methods 135, 193–203. http://
dx.doi.org/10.1016/j.jneumeth.2003.12.016.
Knowlton, R.C., 2006. The role of FDG-PET, ictal SPECT, and MEG in the epilepsy surgery
evaluation. Epilepsy Behav. 8, 91–101. http://dx.doi.org/10.1016/j.yebeh.2005.10.015.
Knowlton, R.C., Razdan, S.N., Limdi, N., Elgavish, R.A., Killen, J., Blount, J., Burneo, J.G.,
Ver Hoef, L., Paige, L., Faught, E., Kankirawatana, P., Bartolucci, A., Riley, K.,
Kuzniecky, R., 2009. Effect of epilepsy magnetic source imaging on intracranial electrode
placement. Ann. Neurol. 65, 716–723. http://dx.doi.org/10.1002/ana.21660.
Kobayashi, E., Bagshaw, A.P., Benar, C.-G., Aghakhani, Y., Andermann, F., Dubeau, F.,
Gotman, J., 2006. Temporal and extratemporal BOLD responses to temporal
lobe interictal spikes. Epilepsia 47, 343–354. http://dx.doi.org/10.1111/j.1528-
1167.2006.00427.x.
Krakow, K., 1999. EEG-triggered functional MRI of interictal epileptiform activity in patients
with partial seizures. Brain 122, 1679–1688. http://dx.doi.org/10.1093/brain/122.9.1679.
Kramer, M.A., Eden, U.T., Kolaczyk, E.D., Zepeda, R., Eskandar, E.N., Cash, S.S., 2010. Co-
alescence and fragmentation of cortical networks during focal seizures. J. Neurosci.
30, 10076–10085. http://dx.doi.org/10.1523/JNEUROSCI.6309-09.2010.
References 29
Kriegel, M.F., Roberts, D.W., Jobst, B.C., 2012. Orbitofrontal and insular epilepsy. J. Clin.
Neurophysiol. 29, 385–391.
Kuzniecky, R., Burgard, S., Faught, E., Morawetz, R., Bartolucci, A., 1993. Predictive value
of magnetic resonance imaging in temporal lobe epilepsy surgery. Arch. Neurol.
50, 65–69.
Lascano, A.M., Perneger, T., Vulliemoz, S., Spinelli, L., Garibotto, V., Korff, C.M.,
Vargas, M.I., Michel, C.M., Seeck, M., 2015. Yield of MRI, high-density electric source
imaging (HD-ESI), SPECT and PET in epilepsy surgery candidates. Clin. Neurophysiol.
http://dx.doi.org/10.1016/j.clinph.2015.03.025.
Laster, D.W., Penry, J.K., Moody, D.M., Ball, M.R., Witcofski, R.L., Riela, A.R., 1985.
Chronic seizure disorders: contribution of MR imaging when CT is normal. AJNR Am.
J. Neuroradiol. 6, 177–180.
Latack, J.T., Abou-Khalil, B.W., Siegel, G.J., Sackellares, J.C., Gabrielsen, T.O., Aisen, A.M.,
1986. Patients with partial seizures: evaluation by MR, CT, and PET imaging. Radiology
159, 159–163. http://dx.doi.org/10.1148/radiology.159.1.3081943.
Le Bihan, D., 2003. Looking into the functional architecture of the brain with diffusion MRI.
Nat. Rev. Neurosci. 4, 469–480. http://dx.doi.org/10.1038/nrn1119.
Lemieux, L., Laufs, H., Carmichael, D., Paul, J.S., Walker, M.C., Duncan, J.S., 2008. Non-
canonical spike-related BOLD responses in focal epilepsy. Hum. Brain Mapp.
29, 329–345. http://dx.doi.org/10.1002/hbm.20389.
Lindquist, M.A., Meng Loh, J., Atlas, L.Y., Wager, T.D., 2009. Modeling the hemodynamic
response function in fMRI: efficiency, bias and mis-modeling. NeuroImage 45, S187–198.
http://dx.doi.org/10.1016/j.neuroimage.2008.10.065.
Malak, R., Bouthillier, A., Carmant, L., Cossette, P., Giard, N., Saint-Hilaire, J.-M.,
Nguyen, D.B., Nguyen, D.K., 2009. Microsurgery of epileptic foci in the insular region:
clinical article. J. Neurosurg. 110, 1153–1163.
Malinowska, U., Badier, J.-M., Gavaret, M., Bartolomei, F., Chauvel, P., Benar, C.-G., 2014.
Interictal networks in magnetoencephalography. Hum. Brain Mapp. 35, 2789–2805. http://
dx.doi.org/10.1002/hbm.22367.
Mamelak, A.N., Lopez, N., Akhtari, M., Sutherling, W.W., 2002. Magnetoencephalography-
directed surgery in patients with neocortical epilepsy. J. Neurosurg. 97, 865–873. http://dx.
doi.org/10.3171/jns.2002.97.4.0865.
Mantini, D., Perrucci, M.G., Cugini, S., Ferretti, A., Romani, G.L., Del Gratta, C., 2007.
Complete artifact removal for EEG recorded during continuous fMRI using independent
component analysis. NeuroImage 34, 598–607. http://dx.doi.org/10.1016/j.neuroimage.
2006.09.037.
Marten, F., Rodrigues, S., Suffczynski, P., Richardson, M.P., Terry, J.R., 2009. Derivation and
analysis of an ordinary differential equation mean-field model for studying clinically
recorded epilepsy dynamics. Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 79, 021911.
http://dx.doi.org/10.1103/PhysRevE.79.021911.
Masterton, R.A.J., Harvey, A.S., Archer, J.S., Lillywhite, L.M., Abbott, D.F., Scheffer, I.E.,
Jackson, G.D., 2010. Focal epileptiform spikes do not show a canonical BOLD response in
patients with benign rolandic epilepsy (BECTS). NeuroImage 51, 252–260. http://dx.doi.
org/10.1016/j.neuroimage.2010.01.109.
Michel, C.M., Lantz, G., Spinelli, L., De Peralta, R.G., Landis, T., Seeck, M., 2004.
128-channel EEG source imaging in epilepsy: clinical yield and localization precision.
J. Clin. Neurophysiol. Off. Publ. Am. Electroencephalogr. Soc. 21, 71–83.
Mikuni, N., Nagamine, T., Ikeda, A., Terada, K., Taki, W., Kimura, J., Kikuchi, H.,
Shibasaki, H., 1997. Simultaneous recording of epileptiform discharges by MEG and sub-
dural electrodes in temporal lobe epilepsy. NeuroImage 5, 298–306. http://dx.doi.org/
10.1006/nimg.1997.0272.
Minassian, B.A., Otsubo, H., Weiss, S., Elliott, I., Rutka, J.T., Snead, O.C., 1999. Magnetoen-
cephalographic localization in pediatric epilepsy surgery: comparison with invasive intra-
cranial electroencephalography. Ann. Neurol. 46, 627–633.
Mitzdorf, U., 1987. Properties of the evoked potential generators: current source-density anal-
ysis of visually evoked potentials in the cat cortex. Int. J. Neurosci. 33, 33–59.
Mohamed, I.S., Gibbs, S.A., Robert, M., Bouthillier, A., Leroux, J.-M., Khoa Nguyen, D.,
2013. The utility of magnetoencephalography in the presurgical evaluation of refractory
insular epilepsy. Epilepsia 54, 1950–1959. http://dx.doi.org/10.1111/epi.12376.
Mori, S., Kaufmann, W.E., Davatzikos, C., Stieltjes, B., Amodei, L., Fredericksen, K.,
Pearlson, G.D., Melhem, E.R., Solaiyappan, M., Raymond, G.V., Moser, H.W., van Zijl,
P.C.M., 2002. Imaging cortical association tracts in the human brain using diffusion-
tensor-based axonal tracking. Magn. Reson. Med. 47, 215–223.
Mormann, F., Andrzejak, R.G., Kreuz, T., Rieke, C., David, P., Elger, C.E., Lehnertz, K.,
2003. Automated detection of a preseizure state based on a decrease in synchronization
in intracranial electroencephalogram recordings from epilepsy patients. Phys. Rev. E
67, http://dx.doi.org/10.1103/PhysRevE.67.021912.
Newey, C.R., Wong, C., Irene Wang, Z., Chen, X., Wu, G., Alexopoulos, A.V., 2013. Opti-
mizing SPECT SISCOM analysis to localize seizure-onset zone by using varying z scores.
Epilepsia 54, 793–800. http://dx.doi.org/10.1111/epi.12139.
Nguyen, D.K., Nguyen, D.B., Malak, R., Leroux, J.-M., Carmant, L., Saint-Hilaire, J.-M.,
Giard, N., Cossette, P., Bouthillier, A., 2009. Revisiting the role of the insula in refractory par-
tial epilepsy. Epilepsia 50, 510–520. http://dx.doi.org/10.1111/j.1528-1167.2008.01758.x.
Niazy, R.K., Beckmann, C.F., Iannetti, G.D., Brady, J.M., Smith, S.M., 2005. Removal of
FMRI environment artifacts from EEG data using optimal basis sets. NeuroImage
28, 720–737. http://dx.doi.org/10.1016/j.neuroimage.2005.06.067.
Nieuwenhuys, R., 2012. The insular cortex: a review. In: Progress in Brain Research. Elsevier,
Amsterdam, The Netherlands, pp. 123–163.
Ormson, M.J., Kispert, D.B., Sharbrough, F.W., Houser, O.W., Earnest, F., Scheithauer, B.W.,
Laws, E.R., 1986. Cryptic structural lesions in refractory partial epilepsy: MR imaging and
CT studies. Radiology 160, 215–219. http://dx.doi.org/10.1148/radiology.160.1.3086931.
Ortega, G.J., Sola, R.G., Pastor, J., 2008. Complex network analysis of human ECoG data.
Neurosci. Lett. 447, 129–133. http://dx.doi.org/10.1016/j.neulet.2008.09.080.
Otte, W.M., van Eijsden, P., Sander, J.W., Duncan, J.S., Dijkhuizen, R.M., Braun, K.P.J.,
2012. A meta-analysis of white matter changes in temporal lobe epilepsy as studied with
diffusion tensor imaging. White matter changes in TLE, Epilepsia 53, 659–667. http://dx.
doi.org/10.1111/j.1528-1167.2012.03426.x.
Park, H.-M., Nakasato, N., Tominaga, T., 2012. Localization of abnormal discharges causing
insular epilepsy by magnetoencephalography. Tohoku J. Exp. Med. 226, 207–211.
Penfield, W., Faulk, M.E., 1955. The insula: further observations on its function. Brain
78, 445–470. http://dx.doi.org/10.1093/brain/78.4.445.
Penfield, W., Jasper, H., 1954. Epilepsy and the Functional Anatomy of the Human Brain.
Little, Brown & Co, Oxford.
Pflieger, M.E., Simpson, G.V., Ahlfors, S.P., Ilmoniemi, R.J., 2000. Superadditive information
from simultaneous MEG/EEG data. In: Aine, C.J., Stroink, G., Wood, C.C., Okada, Y.,
Swithenby, S.J. (Eds.), Biomag 96. Springer New York, New York, NY, pp. 1154–1157.
References 31
Richardson, M.P., 2012. Large scale brain models of epilepsy: dynamics meets connectomics.
J. Neurol. Neurosurg. Psychiatry 83, 1238–1248. http://dx.doi.org/10.1136/jnnp-2011-
301944.
Rodrı́guez-Cruces, R., Concha, L., 2015. White matter in temporal lobe epilepsy: clinico-
pathological correlates of water diffusion abnormalities. Quant. Imaging Med. Surg.
5, 264–278. http://dx.doi.org/10.3978/j.issn.2223-4292.2015.02.06.
Roper, S.N., Levesque, M.F., Sutherling, W.W., Engel Jr., J., 1993. Surgical treatment of par-
tial epilepsy arising from the insular cortex: report of two cases. J. Neurosurg. 79, 266–269.
Rosenow, F., 2001. Presurgical evaluation of epilepsy. Brain 124, 1683–1700. http://dx.doi.
org/10.1093/brain/124.9.1683.
Roth, B.J., Ko, D., von Albertini-Carletti, I.R., Scaffidi, D., Sato, S., 1997. Dipole localization
in patients with epilepsy using the relistically shaped head model. Electroencephalogr.
Clin. Neurophysiol. 102, 159–166. http://dx.doi.org/10.1016/S0013-4694(96)95111-5.
Ryvlin, P., Kahane, P., 2005. The hidden causes of surgery-resistant temporal lobe epilepsy:
extratemporal or temporal plus? Curr. Opin. Neurol. 18, 125–127.
Ryvlin, P., Bouvard, S., Le Bars, D., De Lamerie, G., Gregoire, M.C., Kahane, P., Froment, J.C.,
Mauguière, F., 1998. Clinical utility of flumazenil-PET versus [18 F]fluorodeoxyglucose-
PET and MRI in refractory partial epilepsy. A prospective study in 100 patients. Brain J.
Neurol. 121 (Pt. 11), 2067–2081.
Ryvlin, P., Rheims, S., Risse, G., 2006. Nocturnal frontal lobe epilepsy. Epilepsia 47 (Suppl. 2),
83–86. http://dx.doi.org/10.1111/j.1528-1167.2006.00698.x.
Salek-Haddadi, A., Merschhemke, M., Lemieux, L., Fish, D.R., 2002. Simultaneous EEG-
correlated ictal fMRI. NeuroImage 16, 32–40. http://dx.doi.org/10.1006/nimg.2002.1073.
Salek-Haddadi, A., Diehl, B., Hamandi, K., Merschhemke, M., Liston, A., Friston, K.,
Duncan, J.S., Fish, D.R., Lemieux, L., 2006. Hemodynamic correlates of epileptiform dis-
charges: an EEG-fMRI study of 63 patients with focal epilepsy. Brain Res. 1088, 148–166.
http://dx.doi.org/10.1016/j.brainres.2006.02.098.
Scott, C.A., Fish, D.R., Smith, S.J., Free, S.L., Stevens, J.M., Thompson, P.J., Duncan, J.S.,
Shorvon, S.D., Harkness, W.F., 1999. Presurgical evaluation of patients with epilepsy and nor-
mal MRI: role of scalp video-EEG telemetry. J. Neurol. Neurosurg. Psychiatry 66, 69–71.
Sharon, D., H€am€al€ainen, M.S., Tootell, R.B.H., Halgren, E., Belliveau, J.W., 2007. The
advantage of combining MEG and EEG: comparison to fMRI in focally stimulated
visual cortex. NeuroImage 36, 1225–1235. http://dx.doi.org/10.1016/j.neuroimage.2007.
03.066.
Shiraishi, H., Ahlfors, S.P., Stufflebeam, S.M., Takano, K., Okajima, M., Knake, S.,
Hatanaka, K., Kohsaka, S., Saitoh, S., Dale, A.M., Halgren, E., 2005. Application of mag-
netoencephalography in epilepsy patients with widespread spike or slow-wave activity.
Sijbers, J., Michiels, I., Verhoye, M., Van Audekerke, J., Van der Linden, A., Van Dyck, D.,
1999. Restoration of MR-induced artifacts in simultaneously recorded MR/EEG data.
Magn. Reson. Imaging 17, 1383–1391.
Silfvenius, H., Gloor, P., Rasmussen, T., 1964. Evaluation of insular ablation in surgical treat-
ment of temporal lobe epilepsy. Epilepsia 5, 307–320. http://dx.doi.org/10.1111/j.1528-
1157.1964.tb03338.x.
Spencer, S.S., 1994. The relative contributions of MRI, SPECT, and PET imaging in epilepsy.
Epilepsia 35, S72–S89.
Spencer, S.S., 2002. Neural networks in human epilepsy: evidence of and implications for
treatment. Epilepsia 43, 219–227.
Sperli, F., Spinelli, L., Seeck, M., Kurian, M., Michel, C.M., Lantz, G., 2006. EEG source im-
aging in pediatric epilepsy surgery: a new perspective in presurgical workup. Epilepsia
47, 981–990. http://dx.doi.org/10.1111/j.1528-1167.2006.00550.x.
Srivastava, G., Crottaz-Herbette, S., Lau, K.M., Glover, G.H., Menon, V., 2005. ICA-based pro-
cedures for removing ballistocardiogram artifacts from EEG data acquired in the MRI scan-
ner. NeuroImage 24, 50–60. http://dx.doi.org/10.1016/j.neuroimage.2004.09.041.
Stam, C.J., van Straaten, E.C.W., 2012. Go with the flow: use of a directed phase lag index
(dPLI) to characterize patterns of phase relations in a large-scale model of brain dynamics.
NeuroImage 62, 1415–1428. http://dx.doi.org/10.1016/j.neuroimage.2012.05.050.
Stefan, H., 1993. Clinical applications of MEG in epilepsy. Brain Topogr. 5, 425–427.
Surbeck, W., Gibbs, S.A., Jamali, S., Bouthillier, A., Nguyen, D.K., 2014. Insular cortex ep-
ilepsy: clinical, diagnostic and therapeutic aspects. In: Insula: Neuroanatomy, Function
and Clinical Disorders. Nova main publishers Inc, New York, NY, pp. 105–117.
Tana, M.G., Bianchi, A.M., Sclocco, R., Franchin, T., Cerutti, S., Leal, A., 2012. Parcel-based
connectivity analysis of fMRI data for the study of epileptic seizure propagation. Brain
Topogr. 25, 345–361. http://dx.doi.org/10.1007/s10548-012-0225-2.
Tanaka, N., Cole, A.J., von Pechmann, D., Wakeman, D.G., H€am€al€ainen, M.S., Liu, H.,
Madsen, J.R., Bourgeois, B.F., Stufflebeam, S.M., 2009. Dynamic statistical parametric
mapping for analyzing ictal magnetoencephalographic spikes in patients with intractable
frontal lobe epilepsy. Epilepsy Res. 85, 279–286. http://dx.doi.org/10.1016/j.
Tanaka, N., H€am€al€ainen, M.S., Ahlfors, S.P., Liu, H., Madsen, J.R., Bourgeois, B.F., Lee, J.W.,
Dworetzky, B.A., Belliveau, J.W., Stufflebeam, S.M., 2010. Propagation of epileptic
spikes reconstructed from spatiotemporal magnetoencephalographic and electroencepha-
lographic source analysis. NeuroImage 50, 217–222. http://dx.doi.org/10.1016/j.
neuroimage.2009.12.033.
Taylor, K.S., Seminowicz, D.A., Davis, K.D., 2009. Two systems of resting state connectivity
between the insula and cingulate cortex. Hum. Brain Mapp. 30, 2731–2745. http://dx.doi.
org/10.1002/hbm.20705.
Thornton, R., Laufs, H., Rodionov, R., Cannadathu, S., Carmichael, D.W., Vulliemoz, S.,
Salek-Haddadi, A., McEvoy, A.W., Smith, S.M., Lhatoo, S., Elwes, R.D.C., Guye, M.,
Walker, M.C., Lemieux, L., Duncan, J.S., 2010. EEG correlated functional MRI and post-
operative outcome in focal epilepsy. J. Neurol. Neurosurg. Psychiatry 81, 922–927. http://
dx.doi.org/10.1136/jnnp.2009.196253.
Tousseyn, S., Dupont, P., Goffin, K., Sunaert, S., Van Paesschen, W., 2014. Sensitivity and
specificity of interictal EEG-fMRI for detecting the ictal onset zone at different statistical
thresholds. Front. Neurol. 5, http://dx.doi.org/10.3389/fneur.2014.00131.
Van Mierlo, P., Carrette, E., Hallez, H., Raedt, R., Meurs, A., Vandenberghe, S., Van Roost,
D., Boon, P., Staelens, S., Vonck, K., 2013. Ictal-onset localization through connectivity
analysis of intracranial EEG signals in patients with refractory epilepsy. Epilepsia
54, 1409–1418. http://dx.doi.org/10.1111/epi.12206.
Van Mierlo, P., Papadopoulou, M., Carrette, E., Boon, P., Vandenberghe, S., Vonck, K.,
Marinazzo, D., 2014. Functional brain connectivity from EEG in epilepsy: seizure predic-
tion and epileptogenic focus localization. Prog. Neurobiol. 121, 19–35. http://dx.doi.org/
10.1016/j.pneurobio.2014.06.004.
Varotto, G., Tassi, L., Franceschetti, S., Spreafico, R., Panzica, F., 2012. Epileptogenic net-
works of type II focal cortical dysplasia: a stereo-EEG study. NeuroImage 61, 591–598.
http://dx.doi.org/10.1016/j.neuroimage.2012.03.090.
References 33
Vaugier, L., Aubert, S., McGonigal, A., Trebuchon, A., Guye, M., Gavaret, M., Regis, J.,
Chauvel, P., Wendling, F., Bartolomei, F., 2009. Neural networks underlying hyperkinetic
seizures of “temporal lobe” origin. Epilepsy Res. 86, 200–208. http://dx.doi.org/10.1016/j.
Von Lehe, M., Wellmer, J., Urbach, H., Schramm, J., Elger, C.E., Clusmann, H., 2008. Insular
lesionectomy for refractory epilepsy: management and outcome. Brain 132, 1048–1056.
http://dx.doi.org/10.1093/brain/awp047.
Von Oertzen, T.J., Mormann, F., Urbach, H., Reichmann, K., Koenig, R., Clusmann, H.,
Biersack, H.J., Elger, C.E., 2011. Prospective use of subtraction ictal SPECT coregistered
to MRI (SISCOM) in presurgical evaluation of epilepsy. SISCOM in epilepsy surgery,
Warren, C.P., Hu, S., Stead, M., Brinkmann, B.H., Bower, M.R., Worrell, G.A., 2010. Syn-
chrony in normal and focal epileptic brain: the seizure onset zone is functionally discon-
nected. J. Neurophysiol. 104, 3530–3539. http://dx.doi.org/10.1152/jn.00368.2010.
Watts, D.J., Strogatz, S.H., 1998. Collective dynamics of “small-world” networks. Nature
393, 440–442. http://dx.doi.org/10.1038/30918.
Wendling, F., 2003. Epileptic fast intracerebral EEG activity: evidence for spatial decorrela-
tion at seizure onset. Brain 126, 1449–1459. http://dx.doi.org/10.1093/brain/awg144.
Wendling, F., Ansari-Asl, K., Bartolomei, F., Senhadji, L., 2009. From EEG signals to brain
connectivity: a model-based evaluation of interdependence measures. J. Neurosci.
Methods 183, 9–18. http://dx.doi.org/10.1016/j.jneumeth.2009.04.021.
Westmoreland, B.F., 1998. The EEG findings in extratemporal seizures. Epilepsia 39, S1–S8.
Wiebe, S., Blume, W.T., Girvin, J.P., Eliasziw, M., Effectiveness and Efficiency of Surgery
for Temporal Lobe Epilepsy Study Group, 2001. A randomized, controlled trial of surgery
for temporal-lobe epilepsy. N. Engl. J. Med. 345, 311–318. http://dx.doi.org/10.1056/
NEJM200108023450501.
Wilke, C., van Drongelen, W., Kohrman, M., He, B., 2010. Neocortical seizure foci localiza-
tion by means of a directed transfer function method. Epilepsia 51, 564–572. http://dx.doi.
org/10.1111/j.1528-1167.2009.02329.x.
Winston, G.P., 2015. The potential role of novel diffusion imaging techniques in the under-
standing and treatment of epilepsy. Quant. Imaging Med. Surg. 5, 279–287. http://dx.doi.
org/10.3978/j.issn.2223-4292.2015.02.03.
Wong, C.H., Birkett, J., Byth, K., Dexter, M., Somerville, E., Gill, D., Chaseling, R.,
Fearnside, M., Bleasel, A., 2009. Risk factors for complications during intracranial elec-
trode recording in presurgical evaluation of drug resistant partial epilepsy. Acta Neurochir.
(Wien) 151, 37–50. http://dx.doi.org/10.1007/s00701-008-0171-7.
Wong, C.H., Bleasel, A., Wen, L., Eberl, S., Byth, K., Fulham, M., Somerville, E.,
Mohamed, A., 2010. The topography and significance of extratemporal hypometabolism
in refractory mesial temporal lobe epilepsy examined by FDG-PET: topography and sig-
nificance of extratemporal hypometabolism. Epilepsia 51, 1365–1373. http://dx.doi.org/
10.1111/j.1528-1167.2010.02552.x.
Yun, C.-H., Lee, S.K., Lee, S.Y., Kim, K.K., Jeong, S.W., Chung, C.-K., 2006. Prognostic
factors in neocortical epilepsy surgery: multivariate analysis. Epilepsia 47, 574–579.
http://dx.doi.org/10.1111/j.1528-1167.2006.00470.x.
Zerouali Y., Pouliot P., Robert M., Mohamed I., Bouthillier A., Lesage F., Nguyen D.K.,
Magnetoencephalographic signature of insular epileptic spikes based on functional
connectivity, Hum. Brain Mapp. (accepted).
Zhang, H., Yao, Q., Zhao, X., Jin, X., Wang, C., Guo, H., You, Y., Wang, H., Gao, G., 2008.
A hypermotor seizure with a focal orbital frontal lesion originating in the insula: a case
report. Epilepsy Res. 82, 211–214. http://dx.doi.org/10.1016/j.eplepsyres.2008.06.013.
CHAPTER
Genetic investigations of the

epileptic encephalopathies:
Recent advances
2
C.T. Myers1, H.C. Mefford2,3
3
Corresponding author: Tel.: +1-206-543-9572; Fax: +1-206-543-3184,
e-mail address: hmefford@uw.edu
Abstract
The epileptic encephalopathies (EEs) are a group of epilepsy syndromes characterized by mul-
tiple seizure types, abundant epileptiform activity, and developmental delay or regression.
Advances in genomic technologies over the past decade have accelerated our understanding
of the genetic etiology of EE, which is largely due to de novo mutations. Chromosome micro-
arrays to detect copy number variants identify a genomic cause in at least 5–10% of cases.
Next-generation sequencing in the form of gene panels or whole exome sequencing have
highlighted the role of de novo sequence changes and revealed extensive genetic heterogene-
ity. The novel gene discoveries in EE implicate diverse cellular pathways including chromatin
remodeling, transcriptional regulation, and mTOR regulation in the etiology of epilepsy,
highlighting new targets for potential therapeutic intervention. In this chapter, we discuss
the rapid pace of gene discovery in EE facilitated by genomic technologies and highlight sev-
eral novel genes and potential therapies.
Keywords
Epileptic encephalopathy, Genetics, Whole exome sequencing, De novo mutation
Abbreviations
CNV copy number variation
EE epileptic encephalopathy
1
CTM is a postdoctoral fellow carrying out exome and targeted candidate gene sequencing in epileptic
encephalopathy.
2
HCM is a physician scientist at the University of Washington whose research focuses on gene discov-
ery in pediatric disorders including severe epilepsies.
Progress in Brain Research, Volume 226, ISSN 0079-6123, http://dx.doi.org/10.1016/bs.pbr.2016.04.006

© 2016 Elsevier B.V. All rights reserved.
35
36 CHAPTER 2 Genetic investigations of the epileptic encephalopathies
EEG electroencephalogram
EOEE early-onset epileptic encephalopathy
GGE genetic generalized epilepsy
ID intellectual disability
IS infantile spasms
LGS Lennox-Gastaut syndrome
WES whole exome sequencing
1 BACKGROUND
The epileptic encephalopathies are a group of severe, early-onset conditions charac-
terized by refractory seizures, developmental delay or regression associated with on-
going epileptic activity, and generally poor prognosis (Berg et al., 2010). Some of the
most well-studied EEs include Dravet, Ohtahara, and West syndromes. The genetics
of EE remained elusive for many years; indeed this class of disorders, which usually
occurs in a single individual with no family history, was long thought to be sporadic
or “acquired” rather than genetic. A major advance came with the discovery that de
novo mutations in SCN1A cause Dravet syndrome (Claes et al., 2001). This discov-
ery, along with the identification of CHRNA4 mutations in autosomal dominant
nocturnal frontal lobe epilepsy (Steinlein et al., 1995), KCNQ2 mutations in benign
familial neonatal seizures (Biervert et al., 1998), and others led to the
“channelopathy” hypothesis, which postulates that dysfunction or dysregulation of
ion channels is a common mechanism underlying epilepsy syndromes.
Prior to 10 years ago, gene discovery relied on linkage in large families to nar-
row the search space for disease-causing mutations; even with linkage data, a can-
didate gene approach usually ensued due to the fact that linkage intervals often still
harbored many potential candidate genes. In the absence of linkage data—which is
impossible to obtain in sporadic disorders such as EE—a candidate gene approach
might be the only option. In rare cases, a chromosome abnormality might point to a
candidate chromosomal region.
The emergence of genomic technologies, including chromosome microarrays to
identify copy number variants (CNVs) and next-generation sequencing to identify
single-nucleotide variants (SNVs), over the past decade has accelerated our ability
to scan the entire genome for mutations. These technologies can be applied to an
individual, a family or a large cohort in a hypothesis-driven or an unbiased and
hypothesis-free manner. One of the most notable advances in human genetics facil-
itated by genome-wide mutation screening has been the efficient identification of de
novo pathogenic mutations in sporadic disorders. This is most often achieved by
comparing genome-wide testing results for a patient and both (unaffected) parents.
The application of genomic technologies to patients with EE has helped define
the genetic architecture of these disorders, which are primarily due to de novo mu-
tations. Importantly, the pathogenic mutations identified occur in a surprisingly large
number of genes, highlighting the genetic heterogeneity of EE, and have expanded
2 CNVs in epilepsy 37
our knowledge of critical pathways for epileptogenesis beyond the ion channels,
opening the door for development of novel therapies. In this chapter, we will discuss
the major advances in epilepsy genomics and genetics that have surfaced largely due
to advances in these technologies.
2 CNVs IN EPILEPSY
CNVs are deletions and duplications of stretches of DNA ranging from 1 kb to an
entire chromosome. CNVs are an important source of normal genomic variation,
but some act as risk factors or causes of disease. The development of chromosome
microarrays—which include comparative genomic hybridization and SNP genotyp-
ing arrays—allowed genome-wide screening for CNVs in large cohorts. CNV stud-
ies have been carried out for genetic generalized epilepsy (GGE), focal epilepsy, and
EE, revealing a clear role for CNVs in each major class. Overall, rare CNVs, some of
which involve known disease genes, contribute to 5–10% of cases of childhood ep-
ilepsies (Mefford et al., 2011; Olson et al., 2014).
In GGE, recurrent deletions at three genomic loci have been identified as risk
factors for GGE. Several studies have shown that deletions at 15q11.2, 15q13.3,
and 16p13.11 are significantly enriched in patients with GGE (De Kovel et al.,
2010; Helbig et al., 2009; Lal et al., 2015; Mefford et al., 2010), with the 15q13 de-
letion conferring the most significant risk for epilepsy (odds ratio 68) (Dibbens et al.,
2009). Notably, each deletion exhibits variable inheritance patterns (de novo or
inherited) and incomplete penetrance (Helbig et al., 2013), and all three deletions
are known risk factors for intellectual disability, autism and schizophrenia as well,
highlighting a shared genetic susceptibility for these disorders. The 16p13.11 dele-
tion was also enriched in a large cohort of patients with focal epilepsy (Heinzen
et al., 2010).
In EE, pathogenic CNVs account for approximately 3–5% of cases (Epilepsy
Phenome/Genome Project and Epi4K Consortium, 2015; Mefford et al., 2011),
though deletions of 15q13.3, 15q11.2, and 16p13.11, important for generalized
and focal epilepsies, are rarely seen EE. This is consistent with the notion that EE
has a different genetic architecture than the more common generalized and focal ep-
ilepsies (Mefford, 2014).
In rare disorders, such as EE, identifying regions of overlap among nonrecurrent
deletions in similarly affected patients can point to novel disease genes. This ap-
proach prompted targeted sequencing of CHD2, a gene located in 15q26 deletions,
and led to the discovery that de novo mutations cause an epileptic encephalopathy
characterized by myoclonic seizures, photosensitivity, and developmental delays
(Carvill et al., 2013a; Thomas et al., 2015). Targeted sequencing of SLC6A1, one
of two genes in a minimally deleted region of 3p25 microdeletions, led to the dis-
covery of de novo mutations in 4% of patients with myoclonic astatic epilepsy
(Carvill et al., 2015b). SLC6A1 encodes a GABA transporter responsible for reuptake
of GABA from the synapse. Conversely, the discovery of de novo mutations in
PURA in patients with epileptic encephalopathy helped define PURA as the critical
gene in patients with 5q31.3 deletions (Hunt et al., 2014; Lalani et al., 2014).
3 MASSIVELY PARALLEL SEQUENCING IN EPILEPSY

Next-generation (also called massively parallel) sequencing technology has revolu-
tionized gene discovery. Applications of next-generation sequencing include gene
panel, whole exome, and whole genome sequencing. Gene panels offer simultaneous
sequencing of multiple genes (ranging from several to several hundred), and whole
exome sequencing (WES) refers to sequencing the coding portion of nearly all
20,000 genes in the genome; these two approaches are used most frequently in
the clinic today and offer a rapid, affordable approach to mutation identification.
Whole genome sequencing, in which all 3 billion bases of the genome are se-
quenced, is primarily used in the research laboratory but will inevitably enter the
clinical realm for diagnostics as it becomes more affordable.
Exome sequencing has led to major advances in understanding the genetics of
EE. Because of the severity of disease, cases are usually sporadic with no other af-
fected family members, precluding linkage analysis as a means to gene discovery.
The discovery in 2001 that de novo mutations in SCN1A cause Dravet syndrome
(Claes et al., 2001), one of the most well-studied epileptic encephalopathies, set
the stage for the de novo paradigm for this class of disorders, but it was not until
next-generation sequencing became widely available that the importance of de novo
mutation in non-Dravet EE was solidified. Some of the recent major advances are
discussed later.
In 2012, whole genome sequencing in a single family with a severely affected
child revealed a de novo SCN8A mutation in the proband (Veeramah et al.,
2012b). Since that first report, >40 cases with a de novo mutation in SCN8A have
been reported (De Kovel et al., 2014; Estacion et al., 2014; Larsen et al., 2015a; Ohba
et al., 2014). The clinical presentation includes seizure onset around 5 months of age,
developmental delays, intellectual disability, and movement abnormalities in some
individuals. Mutations in the SCN8A gene appear to cause hyperactivation of the
encoded NaV1.6 sodium channel (Wagnon and Meisler, 2015), and some studies sug-
gest that patients with SCN8A-related EE may respond to the sodium channel blocker
class of antiepileptic drugs (Boerma et al., 2016; Larsen et al., 2015b).
Exome sequencing in 39 patients and follow-up studies in 157 additional individ-
uals with fever-associated epilepsies similar to Dravet syndrome identified a total of
six de novo mutations in HCN1 (Nava et al., 2014b). HCN1 belongs to a family of
hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels that regulate
neuronal excitability. Previous studies suggested that rare variants in HCN1 and
HCN2 are risk factors for GGE (Tang et al., 2008). Another study of 13 unsolved
patients with Dravet syndrome revealed mutations in GABRA1 and STXBP1
(Carvill et al., 2014), two genes previously implicated in other EEs. GABRA1 en-
codes the alpha 1 subunit of the GABAA receptor, a multisubunit chloride channel
3 Massively parallel sequencing in epilepsy 39
that serves as the receptor for the inhibitory neurotransmitter gamma-aminobutyric

acid (GABA). STXBP1 encodes a syntaxin-binding protein that is critical for presyn-
aptic vesicle docking and fusion.
An important advance in our understanding of epilepsy aphasia syndromes came
with the discovery of both inherited and de novo mutations in GRIN2A in affected
individuals (Carvill et al., 2013b; Lemke et al., 2013a; Lesca et al., 2013a). This class
of disorders, which includes Landau-Kleffner syndrome and EE with continuous
spike-waves in sleep, was once hypothesized to be acquired or nongenetic. However
these recent studies confirm that 10–20% of cases are clearly genetic and suggest that
additional genes for the remaining cases are yet to be discovered.
Most of the discoveries described earlier, along with others (Table 1), support the
“channelopathy” hypothesis or epilepsy (Helbig et al., 2008). However, one of the
distinct advantages of genome-wide screening technologies is the ability to use a
hypothesis-free approach and discover the unexpected. In epilepsy, the unexpected
discoveries have included genes involved in chromatin remodeling and transcrip-
tional regulation, synaptic vesicle trafficking, and mammalian target of rapamycin
(mTOR) signaling.
There are now several examples of genes implicated in EE that code for proteins
involved in chromatin remodeling and transcriptional regulation. Using targeted
(gene panel) sequencing of candidate genes located in EE-related CNVs, mutations
in CHD2 were identified in 5/500 patients with EE (Carvill et al., 2013a). CHD2
encodes chromodomain helicase DNA-binding protein 2, a chromatin remodeling
factor. Mutations in CHD2 were also identified through exome sequencing in two
Dravet-like cases (Suls et al., 2013a). To date, more than 20 patients with mutations
in CHD2 have been identified, the majority confirmed de novo events (Carvill et al.,
1993). The epilepsy phenotype is characterized by multiple seizures types, primarily
myoclonic, and exquisite photosensitivity in most patients (Thomas et al., 2015).
Variants in CHD2 have been shown to be a risk factor for photosensitivity in the gen-
eralized epilepsies (Galizia et al., 2015). Notably, mutations have also been reported
in patients with autism (O’Roak et al., 2014) and in ID without seizures (Hamdan
et al., 2014). Careful phenotyping of additional patients with mutations in CHD2 will
help define the phenotypic spectrum, which will undoubtedly be broader than that
described for the first series of patients. Another gene that causes a surprisingly spe-
cific neural phenotype is the transcription factor, MEF2C (Myocycte Enhancer Fac-
tor 2C). While MEF2C has important regulatory roles in other tissue types such as
cardiac and skeletal muscle, it is a critical gene in neural progenitor cell differenti-
ation and maturation, and the causative gene in 5q14 deletion syndrome. Haploin-
sufficiency can cause a range of features including hyperkinesis, variable
epilepsy, intellectual disability, and autism (Paciorkowski et al., 2013), as well as
atypical Rett syndrome (Lambert et al., 2012; Zweier et al., 2010).
EEF1A2, which encodes the alpha-2 subunit of eukaryotic elongation factor-1, is
another gene in which de novo mutations have recently been identified. Four patients
with severe early myoclonic epilepsy, hypotonia, and developmental delay in three
different studies (de Ligt et al., 2012b; Nakajima et al., 2014b; Veeramah et al., 2013)
Table 1 Novel Epilepsy Gene Discoveries from 2012 to 2014

Novel Epilepsy Gene Discoveries
#
Gene Phenotype(s) Casesa References
ALG13 IS, LGS 4 Epi4k Consortium et al. (2013), Michaud

et al. (2014), and de Ligt et al. (2012a)
CERS1 PME 1a Vanni et al. (2014)
CHD2 EOEE, LGS, EE, >20 Paemka et al. (2013), Chenier et al. (2014),
ASD Epi4k Consortium et al. (2013), Lund et al.
(2014), and Suls et al. (2013b)
DEPDC5 FFEVF, ADNFLE, >40 Baulac et al. (2015), D’Gama et al. (2015),
BECTS, FCD, HME Dibbens et al. (2013), Ishida et al. (2013),
Lal et al. (2014), Picard et al. (2014), and
Scheffer et al. (2014)
DNM1 IS, LGS 5 EuroEPINOMICS-RES Consortium (2014)
EEF1A2 IS, EOEE, ASD, ID, 4 Nakajima et al. (2014a), Veeramah et al.
microcephaly (2013), and de Ligt et al. (2012a)
GABRA1 DS, IS, JME, CAE, >10 Carvill et al. (2014), Epi4k Consortium
GGE et al. (2013), Cossette et al. (2002),
Lachance-Touchette et al. (2011), and
Maljevic et al. (2006)
GABRB3 IS, LGS 4 Epi4k Consortium et al. (2013)
GNAO1 OS, IS, EE 6 Nakamura et al. (2013) and
EuroEPINOMICS-RES Consortium (2014)
GRIN2A LKS, CSWS, >50 Carvill et al. (2013c), DeVries and Patel
BECTS, ABPE, EE (2013), Lemke et al. (2013b), Lesca et al.
(2013b), Venkateswaran et al. (2014),
Conroy et al. (2014), and Dyment et al.
(2014)
GRIN2B IS, LGS, FE/ID, ID, >10 Lemke et al. (2014), Epi4k Consortium
ASD et al. (2013), de Ligt et al. (2012a), and
O’Roak et al. (2014)
HCN1 EOEE 4 Nava et al. (2014a)
KCNB1 IS 4 Torkamani et al. (2014) and
EuroEPINOMICS-RES Consortium (2014)
KCNA2 EE 6 Syrbe et al. (2015)
KCNC1 PME 13b Muona et al. (2015)
KCNQ2 BFNS, EOEE, EE >50 Weckhuysen et al. (2012), Richards et al.
(2004), Singh et al. (1998), Singh et al.
(2003), Weckhuysen et al. (2013), and
Allen et al. (2014)
KCNT1 MPSI, ADNFLE 14 Barcia et al. (2012), Heron et al. (2012),
Ishii et al. (2013), and McTague et al.
(2013)
MTOR FCD 18 Lim et al. (2015) and Nakashima et al.
(2015)
NECAP1 EOEE 1a Alazami et al. (2014)
Another random document with
no related content on Scribd:
open window and sniffed the fresh salt breeze appreciatively, and listened to the murmur
of the sea. It seemed a very beautiful world to Peggy in spite of her lack of sight.
CHAPTER V
AN UNEXPECTED VISITOR
IN a very few days, Peggy had settled into the routine of life at Lower Brimley, and had
become well known by sight to the villagers, who took a kindly interest in Mrs. Tiddy's
guest—"the pretty little maid," as they called her, who, though she was blind, could play
the piano, so the servants at the farm reported, and was so clever that wherever she went
once she could go by herself a second time.
Accompanied by the lean, long-legged sheep-dog, she was now a familiar figure on the
beach, where she would sit for hours, listening to the incessant murmur of the sea or
talking to the fishermen, whose deep voices insensibly softened in addressing her. For
nowhere so much as in Cornwall is more respect shown to those whom God has afflicted;
and, though Peggy knew it not, she was continually watched by friendly eyes to see that
she came to no harm.
Mrs. Tiddy, who had been nervous about letting her visitor wander out of her sight at first,
soon grew less vigilant, and was quite satisfied if she knew Wolf was with her, for the dog
had constituted himself her faithful companion, and showed marked signs of jealousy, if
any stranger came near her.
One afternoon, about a week after her arrival at Lower Brimley, Peggy was standing in a
gateway waiting for Mr. Tiddy, who had gone across a meadow to look at some sheep,
when Wolf, as usual at her side, gave a low, warning growl and drew closer to her. She put
her hand on the dog's collar and listened, hearing at length the sound of footsteps slowly
approaching. Some one was evidently ascending the hill which led from the village to the
farm.
After that one growl, Wolf remained silent, and Peggy did not move as the footsteps drew
near. But when they suddenly stopped, the little girl, still holding the dog by the collar,
turned her face, with an inquiring expression upon it, towards the spot where she knew
some one—a woman, she thought, from the sound of the footsteps—to be.
"Can you tell me, if this is the way to Lower Brimley Farm?" asked a somewhat patronising
voice—the voice of a lady, Peggy's sharp ears informed her at once.
"Yes," the little girl answered. "You will come within sight of the house, I know, when you
turn the next corner. Are you going to call on Mrs. Tiddy? Perhaps you are a friend of hers?
She is not at home; she has driven in to Penzance."
"And I have driven from Penzance. But I have no acquaintance with Mrs. Tiddy—the
mistress at the farm, I presume? I have no desire to see her, but I want to have a look at
her flowers. I am told the daffodils and narcissi at Lower Brimley are especially fine. People
talk so much nowadays of the flower-farms of Cornwall that I am curious to see one."
The speaker—a tall, thin, erect old lady, with snow-white hair and very sharp dark eyes,
looked carelessly at the child, and proceeded to question her: "Do you live here? Are you
the farmer's daughter?"
"No; I am no relation to either Mr. or Mrs. Tiddy, although they are so very kind to me,"
Peggy answered simply. "My home is in London; I am only here on a visit. I am sure Mr.
Tiddy will let you look at his flowers; he is very proud of them, and no wonder, for they are
so beautiful! He has gone across the meadow, but he will return directly. Perhaps you can
see him?"
"Do you mean that big man in breeches and leggings?"
"Yes, that's Mr. Tiddy. I promised to wait here with Wolf—that's the dog—till he came back.
Is he far off? Is he coming this way?"
"Cannot you use your eyes, child?" began the lady, a trifle impatiently. Then she paused
abruptly, and scanned the little girl's face with keener scrutiny.
"I cannot see," Peggy responded, "because I am blind."
"Blind! How shocking!"
The stranger's voice had softened perceptibly, and sounded no longer indifferent. Peggy,
conscious of the change, smiled, and a faint colour rose to her pale cheeks as she
remarked:
"Every one is surprised to hear I am blind, but it is quite true."
"And have you been blind long?"
"All my life."
"And yet you look happy!" was the wondering exclamation.
"I am very happy. Mother says I must always remember how many blessings God has
given me, and so I do. Oh, here is Mr. Tiddy!" the little girl cried, with a sudden change of
tone.
The farmer came up, glancing curiously at Peggy's companion, who now put forward a
request—it sounded almost like a command—to see his flowers, adding that she had come
from Penzance on purpose to look at them, and had left her carriage at the foot of the hill.
"You are just in time to see them at their best," Mr. Tiddy told her pleasantly. "In another
week, I shall have cut them all: we rear them for the London markets. Lead the way,
Peggy. A little friend of ours from town," he explained, lowering his voice as the child and
the dog went on ahead. "She's been laid up ill and hasn't picked up her health and spirits
yet. We're trying what our Cornish air will do for her."
"I trust it will do wonders," said the lady, and her voice, though still cold in tone, was not
ungracious. "She looks a delicate child, and she tells me, she is blind."
"Ah, yes, poor dear," sighed Mr. Tiddy. "Though I don't know why I should pity her," he
proceeded, "for she's as happy as the day is long. Her father—he's the organist of St.
John's in the East End of London—calls her 'little Sunbeam,' and the name just suits her.
Her mother and my wife were school friends, and—but here we are!"
The stranger was evidently much gratified by the sight of the flowers, and she was greatly
impressed by the knowledge Peggy evinced concerning them. And the more she conversed
with Mr. Tiddy, the more gracious her manner became, till by-and-by she asked him if
there were comfortable lodgings to be had in the neighbourhood.
"There's a farm higher up the hill, the adjoining farm to this, Higher Brimley it's called—
where they let apartments during the summer months," he replied. "I expect they'd
consider themselves fortunate, if they obtained a lodger as early in the year as this. Ford,
the people are called, and Mrs. Ford is a nice, respectable woman who'd make you very
comfortable."
"You never take lodgers here?" the stranger inquired hesitatingly.
"Never," was the decisive answer. "My wife has plenty of work to do in connection with the
poultry and the dairy, and—to be plain—we like our home to ourselves."
When the lady had gazed her fill at the daffodils, Mr. Tiddy led the way into the garden,
which she declared to be her idea of what a country garden should be. The kindly farmer,
pleased at her admiration for his belongings, thereupon invited her into the house, and
had tea brought into the parlour. "I wish my wife was at home," he observed regretfully,
"but Peggy must play hostess in her place."
"And a very nice little hostess she makes," replied the old lady, her curious gaze upon the
child, who was offering her some of Mrs. Tiddy's home-made cake. "Do you always treat
strangers as you are treating me?" she inquired, turning to Mr. Tiddy again. "I have heard
of Cornish hospitality, but I never believed in it till now. You don't know anything about me
—" She paused and laughed rather bitterly, then added: "Most people would not think it
worth while to entertain a stranger—one never likely to cross their path in life again."
"Then you do not mean to seek lodgings in the district?" Mr. Tiddy asked gravely.
"I have not made up my mind on that point yet. I almost think I could be contented in a
spot like this."
Having finished her tea, she rose and prepared to depart. Mr. Tiddy now noted for the first
time, how costly was her dress—evidently she was a woman rich in this world's goods—
and he thought as he glanced at the deep lines of discontent around her hard mouth, that,
in spite of her undeniably handsome face, she was the most ill-tempered looking old lady
he had seen for many a long day, and doubted much if she would be contented anywhere.
"Good-bye, child," she said stretching out her delicately-gloved hand to Peggy. "It is quite
possible that we may meet again."
"If we do, I shall remember you," was the grave response. "I shall remember you by your
voice. And I can't help thinking that somewhere we have met before, or perhaps it is only
that you remind me of some one—that must be it."
The lady looked at Peggy searchingly, and shook her head. Then she went away, leaving
the little girl in a very thoughtful frame of mind. When Mr. Tiddy returned, after having
accompanied the stranger down the hill and placed her in the hired carriage in which she
had been driven from Penzance, he asked Peggy what she thought of their late visitor.
"She seemed rather unhappy, didn't she, Mr. Tiddy?" she questioned.
"Unhappy?" he said, reflectively. "I don't know about that. To me she appeared simply
discontented. She is a selfish woman, I'll be bound—so maybe you're right, my dear, for
selfish folk are never happy—and wrapped up in her own concerns. But she liked my
daffodils, didn't she? I could see she had a real love for flowers. And she was interested in
you, too. One mustn't judge by appearances altogether—"
"I judge by her voice," said Peggy, as he broke off, leaving his sentence unfinished.
"A hard, cold voice, wasn't it?" questioned Mr. Tiddy.
"Y-e-s. Was she very old, Mr. Tiddy?"
"Over seventy, I should say."
"That's a great age, isn't it? I wonder if she is always alone like she was to-day. Perhaps
she has no one to love and care for her now she is old. How sad that must be! Poor old
lady!" And there was deepest sympathy in her tone.
Mr. Tiddy looked at the speaker with a tender smile; but he did not think it worth while to
say that, to him, their visitor had appeared anything but poor. Perhaps, he reflected, the
child might be right after all, for he knew how often those rich in worldly possessions are
poor in heart.
CHAPTER VI
MISS LEIGHTON'S DISCOVERY
THE daffodil blooms had all been gathered; March had given place to April; and, day by
day, Peggy was improving in health, whilst roses—faint as yet, it is true—were appearing in
her cheeks. The doctor's prescription of a change of air was evidently what she had
needed; and Mrs. Tiddy was much gratified at being able to write most cheering reports of
her visitor's condition to Mrs. Pringle, who read them aloud to her husband and Billy with
deep thankfulness in her heart.
"How we shall miss the child when she leaves us!" Mr. Tiddy remarked to his wife one
evening, as they strolled up and down the path in front of the house when the work of the
day was over, listening to the music which Peggy's fingers were drawing from the piano in
the parlour. The little girl was naturally musical and had been well taught by her father,
who had often told her that if she worked hard and practised industriously, she might
become a real musician some day, and to be a real musician was her most earnest desire.
"But she is not going to leave us for a long while yet," Mrs. Tiddy responded. "I have
written and told her mother that she must spare her to us for another month, at least, and
I think she will be glad to let her stay, as her health is benefiting so much by our Cornish
air. By the way, Ebenezer, have you heard that there are lodgers at Higher Brimley? No? An
elderly lady and her maid have taken Mrs. Ford's apartments. They were pointed out to me
in the village this afternoon when Peggy and I were returning from the beach. And Peggy
says she is sure the lady is the one who came from Penzance on purpose to look at our
flowers. She is a tall, thin, old lady with quite white hair."
"You don't say so!" exclaimed the farmer. "I told her she could get apartments at Higher
Brimley, but I did not think she really meant to see about them. Did she speak to Peggy?"
"No; she did not see her, for we were in the post office when she passed with her maid.
Peggy recognised her by her voice."
"I wonder who she is. You did not hear her name, I suppose?"
"No. Listen! The child is singing!"
They stood silently by the open window of the parlour and listened as the little girl's voice,
low and sweet in tone, rang out clearly and softly:
"Holy Father, cheer our way

With Thy love's perpetual ray:
Grant us every closing day
Light at evening time."
"Dear child," murmured Mrs. Tiddy, tears springing involuntarily to her eyes, "it does seem
hard lines that one naturally so bright and joyous should be blind! But there, God knows
best, and I suppose He has denied her sight for some good reason; and she has His love to
cheer her way, I'm certain."
"I think there's light in her heart," said Mr. Tiddy simply, and his wife agreed.
It was on the following morning that Peggy, who had wandered down to the beach with
Wolf in attendance, met the lodgers from Higher Brimley. The old lady spoke to the little
girl, and inquired if she remembered her. And, receiving an answer in the affirmative, she
dismissed her maid, telling her to wait within sight, and requested Peggy to sit down by
her side on an upturned boat, and talk to her for a while.
Peggy complied readily, for she was of a very sociable disposition, and commenced the
conversation by informing her companion that she had recognised her voice when she had
heard it on the previous day.
"I was in the post office with Mrs. Tiddy when you passed," she said, "and you were
talking. We were told you had taken Mrs. Ford's rooms."
"I do not know how long I shall remain there-perhaps only a few days, perhaps longer. I
suppose the daffodils are all gone now?"
"Yes," Peggy nodded regretfully; "but there are more flowers than ever in the garden, and
those will not be cut. Mr. Tiddy grows them for himself and his friends; but the daffodils
and narcissi, he sells."
"You are looking better than when I saw you before," observed the stranger. "I suppose
you will be going home soon?"
"Not for some weeks yet. Oh, yes, I am a lot better! I feel really well; and Mrs. Tiddy says
I am getting quite rosy and sunburnt. I am so glad, because they will be pleased at home."
"Are you one of a long family?"

"No. I have only one brother—Billy. Father is the organist of St. John's, but I do not expect
you know the church. Mr. Maloney is our Vicar. He's a great friend of ours. I'm sure you'd
like him, because he's such a good man. Mother says he's very clever, and people come a
long distance often to hear him preach, so I suppose he must be."
"I think I've heard of him," said the old lady thoughtfully. "He gives up his life to working
amongst the poor, does he not?"
"Yes. Nearly every one in our parish is poor. Mr. Maloney is, I believe, and we are, you
know, because father's salary isn't much, and his music pupils don't pay him as they
ought. But father is very clever, too, and some day I dare say we shall be better off. Father
composes music, and there are very few people who can do that," the little girl said, with a
ring of affectionate pride in her voice. "Do you live in London, too?" she inquired, thinking
it was her turn to ask a question now.
"I have a house in town. Will you come and spend a day with me there when we both go
back to our own homes?"
"I—I hardly know," Peggy replied doubtfully, flushing with surprise. "It's very kind of you to
invite me; but I must ask mother. I don't know who you are, and—"
"And I don't know who you are, either! Suppose you tell me your name?"
"It is Margaret Pringle; but I am always called Peggy, because father calls mother
Margaret."
"Pringle!" exclaimed the old lady, growing suddenly crimson. She looked almost angrily at
Peggy as she spoke, but of course the little girl was unconscious of that fact, though she
caught the sound of agitation in her voice. "Pringle!" she repeated. "Is it possible? Tell me,
is your father's name John?"
"Yes. You have heard of him?" Since her companion had evinced some knowledge of the
Vicar of St. John's, it did not occur to Peggy as at all unlikely that she should know
something of the organist too. "He plays most beautifully," she continued impressively. "Mr.
Tiddy will tell you so, for he heard him one Sunday evening when he went to church with
us. It was the first time I had been to church after my accident. Oh, I haven't told you
about that! I was knocked down when I was out with Billy, and it was a great wonder that I
was not killed!"
And she recounted the story of her adventure at some length, utterly unconscious of the
effect it was having upon her listener, who had lost all her colour again now, and was
looking paler than before.
"The—the person in the carriage would not have understood that you were blind," the old
lady remarked at length, subsequent to a long pause which had followed the conclusion of
Peggy's tale.
"No, of course not," the little girl agreed, "but Mr. Maloney says the least she could have
done would have been to have driven me home. Billy thinks she didn't care, if I was hurt
or not. And—isn't it strange?—she's supposed to be a very charitable person!"
"Then you know who she is?"
"Oh, yes! She gave the policeman her card, and mother used to know her quite well—
years ago."
"Ah!"
"I—I am afraid I have been talking too much," Peggy said hesitatingly, with a sudden touch
of reserve in her tone as she became aware that she had let her tongue run away with her.
She hoped she had not wearied her companion with her chatter.
"Why did you say she—the person in the carriage, I mean—is supposed to be very
charitable?" asked the old lady presently.
"Because she gives away heaps and heaps of money," was the prompt reply.
"Well, that is very generous of her, is it not?"
"Yes. But I don't think she can be really charitable, if she isn't kind in little ways and if
she's unforgiving. I asked Mr. Maloney what he thought."
"Well? What did he say?"
"He repeated that verse in the Corinthians, 'Though I bestow all my goods to feed the
poor, and though I give my body to be burned, and have not charity, it profiteth me
nothing.' He didn't say anything but that; but I know what he meant, don't you? But, don't
let us talk about her any more—I am not sure that I ought to have spoken of her at all."
"You have done no harm. So that accident was actually the cause of your illness?"
"Yes. And just when the doctor said I must have a change of air, Mr. Tiddy arrived and
invited me here. Wasn't it kind of him, and of Mrs. Tiddy too? You know I couldn't possibly
have had a change but for them, for father couldn't have managed it, and it made him so
dreadfully unhappy that he couldn't. Both he and mother were so worried about me."
Soon after that the old lady rose, remarking that she found the wind a trifle chilly. She said
good-bye to Peggy and joined her maid with the intention of returning to her lodgings. Left
alone, the little girl reflected that her late companion had been decidedly less affable at the
conclusion of their conversation, than at the commencement, and wondered why that had
been. Had she unwittingly said anything to cause her annoyance? She felt puzzled and
uneasy; and, though she had been encouraged to talk, she wished she had been less
communicative.
Meanwhile the old lady, who, as the reader has no doubt guessed, was no other than Miss
Leighton, Mrs. Pringle's aunt, was walking up the hill towards Higher Brimley in anything
but a happy frame of mind. That morning she had spoken of remaining some while longer
in Cornwall, and had professed herself quite satisfied with the arrangements which had
been made for her comfort; but now, she had almost decided to quit the neighbourhood at
once.
She had been greatly attracted by the blind child on the occasion of her visit from
Penzance to look at Mr. Tiddy's flowers. And when she had caught sight of her on the
beach an hour previously, she had determined to cultivate her acquaintance. But having
learnt that Peggy was the daughter of the niece whom she had never forgiven for what she
called her ingratitude, she was experiencing mingled feelings of anger, bitterness, and
regret.
"I will have no more to do with her," she thought.
Then she shuddered as she reflected on the accident. How terrible it would have been if
her horses had killed Margaret's little daughter! She had made no inquiries concerning her
niece since her marriage and had not even known where she was living, or if she had any
children or not. Therefore, it had been somewhat of a shock to discover she had a child
who was afflicted with blindness. She pictured Peggy, golden-haired and sunny-faced, and
an unwonted expression of tenderness crossed her countenance. After all, she decided,
she would remain at Higher Brimley for the time, for—it was weak of her, no doubt—she
felt she must see Peggy once again.
CHAPTER VII
A GREAT SURPRISE
NOT quite a week later, Mr. Tiddy, crossing the fields in his usual leisurely fashion towards
the house at dinner-time, caught sight of his wife and Peggy, standing at the garden gate,
evidently waiting for him. As he drew near enough to see the expression of their faces, he
noticed that both appeared excited, and as he joined them the little girl cried eagerly:
"Oh, Mr. Tiddy, we've had a visitor! She came and knocked at the door and asked if she
might go round the garden. And who do you think she was?"
"Why, the old lady who's lodging at Higher Brimley, to be sure," answered Mr. Tiddy
promptly, evincing no surprise. "I met her this morning, and she stopped and spoke to me.
She expressed a desire to see our flowers, so I told her, she'd be welcome to look at them,
whenever she pleased. She didn't lose much time in taking me at my word," he concluded,
smilingly.
"Ah, but do you know who she is?" demanded Peggy. "No, we thought not. You'll be simply
astounded when you hear. She didn't tell us until just as she was leaving, and then she
said her name was Leighton, and that I was related to her—distantly related, she said.
She's mother's Aunt Caroline, the rich lady who was in the carriage when—"
"What!" broke in the farmer, "You don't say so!" He looked questioningly at his wife as he
spoke, and she hastened to reply:
"Yes, Ebenezer, it is true. There can be no doubt about it. She is that rich Miss Leighton of
whom we have heard so much."
"I told her who I was that day she talked to me on the beach," Peggy said, with face and
voice full of excitement. "She asked me my name; and—and I told her, too, all about my
accident and how unkind we thought it of her to have driven away when I was hurt. I think
perhaps she was cross at what I said, but I never dreamt who she was, so I don't think
really it was my fault, do you, Mr. Tiddy?"
"No, my dear, I do not," he agreed.
"Still, perhaps I ought not to have talked as I did to a stranger. She was very nice to-day,
though, wasn't she, Mrs. Tiddy?"
"Very. Will you run into the house, Peggy, and say we are ready for dinner?"
Then as the little girl obeyed, Mrs. Tiddy turned to her husband and said gravely:
"Ebenezer, what can have brought Miss Leighton here? Until Peggy told her her name the
other day, she had no idea who she was or even that her niece had children. I don't
believe she has forgiven Peggy's mother yet. Isn't it shocking to bear malice in one's heart
so long? 'I don't wish to hear anything concerning your friend or her husband,' she said to
me in a tone without an atom of feeling in it; 'but I was never one for visiting the sins of
the parents upon the children. My niece proved herself ungrateful, and I regard ingratitude
as a sin, but I feel no resentment against her innocent daughter.' I should think not
indeed! I made no answer, however, for I was afraid, if I did, I might say too much."
"Surely she did not make that remark before Peggy!" exclaimed Mr. Tiddy, his ruddy colour
deepening with indignation.
"No, certainly not; Peggy was not within hearing then. What shall I do? Miss Leighton
asked me to call on her and bring Peggy with me, and I half promised I would; I did not
like to refuse. I think the old lady has taken a fancy to the child. Isn't it strange that those
two should have crossed each other's path again?"
The farmer nodded, a very thoughtful expression on his face. "There's One above Who
planned they should meet, that's my opinion," he said gravely; "and I don't think we ought
to try to keep them apart. Maybe the old lady will get to feel more kindly towards her niece
when she knows Peggy better and realises what a dear little soul she is and how well her
mother has brought her up. I am sure Mrs. Pringle will not object to your taking the child
to call on her aunt. By the way, does Miss Leighton like her lodgings?"
"She said they were fairly comfortable. She strikes me as a rather dissatisfied body. She is
anything but a happy woman, Ebenezer, though God has given her so much; and I hear
from the servants, who have become friendly with her maid, that she is a very jealous,
exacting temper, and she is always imagining people are trying to cultivate her
acquaintance on account of her wealth."
"Well, she cannot possibly imagine that about you," Mr. Tiddy replied, "for she has sought
your acquaintance herself. I suppose we had better go in to dinner now. There's Peggy
under the porch beckoning to us."
Mrs. Tiddy decided she would not call upon Miss Leighton until she had mentioned the
matter to her old school friend; so she wrote to her that same day, and received an answer
by return of post. Mrs. Pringle said very little about her aunt in her letter, but she raised no
objection to her little daughter's calling with Mrs. Tiddy at Higher Brimley. "Aunt Caroline is
not fond of young people," she remarked, "so please don't force the child upon her notice
—but I am sure you will not do that."
"I certainly will not," Mrs. Tiddy reflected as she folded up her friend's letter, "but I will
take Peggy to call on Miss Leighton, as the old lady made a point of my doing so. We need
not stay very long, any way."
Peggy experienced a feeling of unusual shyness when, one April afternoon, she
accompanied Mrs. Tiddy to Higher Brimley; and, although Miss Leighton received them
with every sign of cordiality, she was anything but at ease in her presence. As the little girl
sat in silence listening to the conversation of the two ladies, she was aware that the elder's
eyes were upon her, and she alternately flushed and paled as she thought over the small
amount of information she had gleaned from her mother since her accident about this aunt
of hers. Her tender heart had gone out in sympathy towards the old lady, whom she had
sincerely pitied because she had fancied she might be all alone in the world, but now she
mentally regarded her from quite another point of view.
"Mother would have loved her, if she would have let her," she reflected. "It is her own fault
if she is lonely. I wonder if she will speak of mother to me!"
But Miss Leighton did not once mention her niece's name. She addressed herself very
kindly to Peggy every now and again, and seemed wishful to make much of her, and Mrs.
Tiddy saw she was disappointed and half-vexed by the child's evident disinclination to talk.
"What have you done with your dog this afternoon?" Miss Leighton inquired, when at
length her visitors rose to go.
"We shut him up in the stable before we started," Peggy answered. "He wanted to come
because he loves a walk."
"He is rather quarrelsome with other dogs," Mrs. Tiddy explained, "so we thought it wiser
to leave him at home. The poor creature was very disappointed, for he spends most of his
time with Peggy now, and we always feel she is safe if Wolf is with her."
"What will he do when he loses her altogether?" asked Miss Leighton. "Peggy does not
propose taking him back with her to London, I presume?"
"No," the little girl answered, accepting the question seriously, "I wouldn't do that, even if
Mr. Tiddy would give him to me, for I am sure he would be wretched in town. I'd rather
know he is here, guarding the yard and looking after the sheep, and going on as he always
does—having such a good time! He will miss me at first, but—where is Mrs. Tiddy?" she
asked quickly.
They had left the house and were in the garden now, Mrs. Tiddy having lingered at the
door to exchange a few words with Mrs. Ford.
"She is talking to my landlady," Miss Leighton replied. "She will be here presently. Are you
in a great hurry to go? You have no objection to being alone with me for a few minutes, I
suppose?" she questioned sharply.
"Oh! No!" Peggy assured her. "And—and now we are alone, I should like to say that I hope
I wasn't rude to you the other day on the beach," she proceeded, looking distressed. "I
would not have spoken like that if—if I had known who you were. I—I have thought of it
often since, and I am sorry if I said anything you did not like. I was afraid, afterwards, that
you were displeased with me."
"People are seldom pleased to hear others' opinions of themselves," was the dry response.
"You evidently considered my conduct towards you had been heartless; but I am not angry
with you, child. You only said frankly what you thought."
"Yes," Peggy agreed, colouring hotly in her confusion. "I am glad you are not angry,
though, because I did not mean to be rude, and I am afraid I must have been," she added
deprecatingly.
"I think you are prejudiced against me." Miss Leighton paused momentarily, and sighed,
then continued, "Well, it is natural you should be. I am sorry, nevertheless. Cannot you
dismiss all you have heard of me from your mind and take me as you find me?"
"I—I will try. I have not heard much about you, indeed! I never heard of you at all till after
my accident! Then Billy told me who you were, and I was so surprised! Billy and I have
often talked of you since!"
"Really? I dare say you heard Mrs. Tiddy ask me to take tea with her one afternoon, soon?
I shall hope then to hear you play. I hear you are quite a musician."
"Oh, no! But I love music. I play to Mr. and Mrs. Tiddy every night." The mantle of reserve
was falling from Peggy and the brightness was returning to her face. "Do you love music
too?" she inquired, lifting her sightless blue eyes to her companion's countenance.
"Indeed I do; so we have that much in common, at any rate."
"Oh, we have more than that, for I am sure you love flowers, and so do I. Do you know,
there are such a lot of sea-pinks growing on the cliffs—"
"You do not go on the cliffs alone?" Miss Leighton interposed.
"Oh, no! But I have been several times with Mr. Tiddy, and I hold fast to his hand. There is
a sheep-track along the cliffs, you know, and it is quite safe if you keep to that. I could
find my way alone, I am sure, but I never mean to try, because I have promised, I won't."
"That's well. Perhaps you and I might walk there together some day. Do you think you
could put up with an old woman for a companion?"
"Yes, Miss Leighton," Peggy answered, smiling.
"And you shall show me the sea-pinks, and we will take Wolf to protect us both. But do not
call me 'Miss Leighton,' child; call me 'Aunt Caroline,' for you are my great-niece and—and
I should like to be kind to you."
CHAPTER VIII
CONCERNING ELLEN BARNES
MISS LEIGHTON'S maid—Ellen Barnes—was a plain, sad-faced, middle-aged woman who

had been with her present employer for many years. She had known Mrs. Pringle before
her marriage, and consequently, it was with considerable satisfaction and some
astonishment that she saw the interest with which her mistress regarded the daughter of
the niece, the very existence of whom she had ignored so long.
It cannot be said that Miss Leighton was on anything like confidential terms with her maid;
but she trusted her, and she would have certainly been at a loss without the services of the
quiet, rather spiritless woman who rarely spoke except in answer to a question.
Miss Leighton had now been nearly a fortnight at Higher Brimley, and had had several
interviews with her little great-niece on the beach, and had walked with her along the
sheep-track on the cliffs to look at the sea-pinks. But she had not yet taken tea with Mrs.
Tiddy as had been suggested, and when, one sunshiny morning, Peggy arrived with an
invitation for her to do so that afternoon, she accepted it immediately.
"Of course I will come," she replied, after Peggy—rosy with the exercise of walking—had
delivered her message. "Please give my kind regards to Mrs. Tiddy and say I accept her
invitation with pleasure. Did you walk here by yourself, child?"
"No," answered Peggy. She had been ushered into Miss Leighton's sitting-room by Ellen
Barnes, who had been on an errand to the post office for her mistress and had overtaken
the little girl on her way home. "I started to come alone," she said, "but I had not gone far
before I heard some one calling to me. It was Barnes. So we walked on together. What a
very nice woman she is, Aunt Caroline! We had such a long talk!"
"Humph!" exclaimed Miss Leighton, rather surprised. "And, pray, what did you find to talk
about?"
"Oh, about things at home, first of all," was the somewhat vague response. "My home, of
course I mean. I did not know till to-day that Barnes knew my mother."
The little girl had taken the chair which had been placed for her close to the open window
by which Miss Leighton was sitting, and the bright spring sunshine fell full upon her face
framed in its golden curls. Certainly she made a very pretty picture.
"I like Barnes," she proceeded in a tone of decision as her companion vouchsafed no
response. "How very fortunate you are to have such a nice woman for your maid, Aunt
Caroline!"
"I believe she is thoroughly trustworthy," Miss Leighton remarked, somewhat astonished at
this expression of opinion, "and that is a great deal to be able to say of any one. Barnes
has been with me many years. I pay her good wages and she is not overworked. I believe
she values her situation."
"Oh, yes, I am sure she does!" Peggy agreed earnestly.
"How can you tell, child?" Miss Leighton asked, a slightly amused smile curving her lips.
"She told me she did, Aunt Caroline."
"Did she?" There was gratification in the old lady's voice. "But—how strange of her to say
so to you! She must have been very confidential."
"She was telling me about her brother, and how she values her situation with you because
you pay her such good wages that she is able to send home more than half she earns. Oh,
Aunt Caroline, when she told me about her brother, I thought how thankful I ought to be
that God has only made me blind! Suppose I was like poor Barnes's brother: how much
worse that would be!"
"What about Barnes's brother?" inquired Miss Leighton, in utter bewilderment. "I have
never heard anything about my maid's relations; she has a week's holiday every summer;
I suppose she goes to see them then. Stay—I think I remember hearing her once mention
a mother, who, by the way, must be a very old woman, for Barnes herself is quite middle-
aged."
"Barnes's mother is more than eighty years old, and she lives in a little village near
Plymouth with her son. Oh, Aunt Caroline, he is only two years younger than Barnes, and
he has been an idiot all his life!"
"Dear me!" exclaimed Miss Leighton, feeling really shocked. "I never heard that before.
Barnes never told me."
Peggy looked intensely surprised for a minute, then an expression of comprehension
crossed her face. "I expect she did not like to tell you," she said. "Perhaps she thought you
would not be interested, you know."
"Why should she think that?" Miss Leighton questioned sharply.
The little girl was silent. She had heard Mrs. Tiddy say that Barnes looked a broken-
spirited woman; and Mrs. Ford, when she had called at Lower Brimley a few days
previously, had declared her to be a perfect slave to her mistress's whims, and wondered
why she did not seek another situation with some one who, at any rate, would be less
inconsiderate and exacting. In the conversation the little girl had had with Barnes, she had
discovered the reason which induced her to keep her post. It was because it enabled her to
do so much for her poor mother and her imbecile brother in their cottage home.
"Why should you think that?" Miss Leighton persisted. "Come, speak out, child! Don't be
afraid of me!"
"I'm not," Peggy answered truthfully, for she was not in the least in awe of the old lady. "I
meant that—that perhaps if you have never asked Barnes about her relations, she would
think you would not care to hear about them. But it does seem so very odd that she
should have lived with you so many years, and you should not know all about her mother
and brother!"
"The brother is an idiot, you say?"
"Yes; but Barnes and her mother are very fond of him; it would break her mother's heart
to be parted from him, and Barnes says they shall never be separated as long as God gives
her health and strength to work. They get parish pay, and with what Barnes sends them
they manage to live pretty comfortably. Oh, Aunt Caroline, mustn't it be dreadful to have a
brother like that! Oh dear, I do think it is so very sad!" And the pitiful tears rose to Peggy's
blue eyes and ran down her cheeks.
"You mustn't take other people's troubles to heart like that!" Miss Leighton exclaimed
hastily.
"I feel so sorry for Barnes," Peggy said, with deepest sorrow in her tone, "because I am
sure it must make her very unhappy to think of her brother and her old mother
sometimes. She must wish to see them so much, and always be wondering how they are
getting on. Mrs. Tiddy says Barnes looks a very sad woman. I wish I could do something to
make her happier."
"I said so to her just now," she continued, with a brightening face, "and what do you think
she answered? That I had helped her by being sorry for her brother; she said she wouldn't
have told me anything about him if I hadn't been afflicted myself, and it warmed her heart
to know I cared. I told her I should pray to God every night to make her brother right in
his mind, and she said she was afraid that would never be in this world. Poor fellow! He's
like me, Aunt Caroline, in that way, isn't he? He will have to bear his cross as long as he
lives, and his cross is so much heavier than mine."
A silence followed, during which Miss Leighton sat gazing, unseeingly, out of the window.
There was a mist before her eyes, and a lump in her throat which prevented her uttering a
word. By-and-by Peggy rose to go.
"Mrs. Tiddy said she hoped you would come early this afternoon," she observed. "Please
do, for I've so many things to show you."
"I certainly will," Miss Leighton replied. "Shall Barnes take you home?"
"Oh no, thank you, I know the way quite well; I have only to keep to the road. Good-bye,
Aunt Caroline—till this afternoon."
Miss Leighton stood at the window and watched the little girl out of sight, a gentler
expression than usual on her face. Then she resumed her seat and took up the book she
had been reading before the child's arrival; but it failed to interest her now, for her mind
was full of uneasy thoughts. Barnes had lived with her for nearly twenty years, she
reflected; and yet how little she really knew of the woman! Well, it could not be expected
that she would interest herself in her maid's concerns. And yet, how surprised Peggy had
been at discovering her ignorance of aged mother and her imbecile son. Peggy had learnt
all there was to know about them in less than half an hour.
Miss Leighton paid her servants liberal wages—she was never stingy where money was
concerned—and it had often occurred to her that Barnes must be of a miserly disposition,
for she dressed very plainly and it had been impossible not to notice that she begrudged
spending money. Now she understood where the woman's wages had gone. Barnes had
not been making a purse for herself, but spending it upon those dear to her, and, all the
while, she had been regarding her as a mean, poor-spirited creature.
It was difficult to realise that the humble, silent woman who had borne with her mistress's
haughty temper so patiently, had been leading a life of self-sacrifice and self-repression
from the noblest of motives; but Miss Leighton now realised that such had been the case,
for Peggy had thrown a new light upon the maid's character.
What had made Barnes tell Peggy about her brother? the old lady wondered. Was it
because her heart had been hungry for sympathy, and she had known instinctively that
she would receive it from the blind child? Probably so. She had preferred to confide in a
stranger, rather than in the mistress whom she knew to be accounted a charitable woman
—one lavish in giving of her wealth.
"I don't think she can really be charitable, if she isn't kind in little ways," Peggy had said
ingenuously, passing her childish judgment on her mother's rich aunt, and the words
returned forcibly to Miss Leighton's mind now, and cause her a strange pang, whilst she
asked herself if she had ever been really kind to Ellen Barnes, or for that matter, to any
member of her household. She was a lonely old woman; but, after all, was it not greatly
owing to her own fault? She had certainly never been "kind in little ways."
CHAPTER IX
TEA AT LOWER BRIMLEY
IT was not the ordinary "afternoon tea" to which Miss Leighton was invited, but a
substantial meal laid on the square mahogany table in the parlour at Lower Brimley, with a
mass of primroses in the centre intermingled with sprays of beautiful fern moss,
surrounded by plates of daintily cut bread-and-butter and various kinds of preserves in
glass dishes, an old china bowl full of clotted cream, a plum cake, and some saffron buns
—"knobbies" as they are called in Cornwall.
It was but natural that Mrs. Tiddy should put her best possessions before this relation of
her little visitor's, so the silver tea-service had come out of its flannel wrappings, and Miss
Leighton drank her tea from a rare old china teacup with a wreath of pink roses inside its
brim—one of a set which had been treasured in Mr. Tiddy's family for three generations
and was only used on great occasions—and stirred her tea with an apostle spoon, worn
thin with age; whilst, much to her hostess's gratification, she evidently appreciated the
efforts which were being made to entertain her.
Seated at Mrs. Tiddy's right hand at the tea-table, the old lady looked about her with a
sense of unusual contentment. For once in a way, she was satisfied with the company in
which she found herself. Yes, she liked this hearty, out-spoken west-country farmer and his
pleasant, intelligent wife, for she was under the impression—a true one—that they would
have welcomed her as cordially if she had been poor instead of rich, and she so seldom felt
that about people. After tea, Peggy took possession of her, and, after visiting the yard and
inspecting the poultry, she was led into the great farm kitchen, where, in one corner of the
oak settle close to the fire was a flannel-lined basket containing two weakly chicks.
"Mrs. Tiddy thought this morning that they would die," Peggy said as she covered the
invalids with her warm hands. "But they are getting on nicely now, and to-morrow, they'll
be strong enough to run with their brothers and sisters."
Miss Leighton glanced around the kitchen with admiring, appreciative eyes, noticing the
shining tins on the mantel-piece, the big copper warming-pan and the tall, brass-faced
clock against the wall, and the linen bags hanging from the beams which spanned the
ceiling, containing home-cured hams and sides of bacon. And then, after a visit to the
dairy, she returned with Peggy to the parlour, where the tea-things had disappeared from
the table, and the easiest chair in the room was drawn near the window for the guest.
"What a peaceful scene it is!" Miss Leighton exclaimed, as her eyes rested on the village
below and the distant sea. "I suppose, Mrs. Tiddy, you have become greatly attached to
this charming spot?"
"Yes," Mrs. Tiddy answered. "I love Lower Brimley as I imagine only a woman who has
been homeless and dependent can love her home. There was nowhere in the world where I
could feel I had a right to be, till I married, for I was left an orphan at an early age and
brought up by relations who regarded me in the light of an incubus. The bread of charity is
very bitter, Miss Leighton—how bitter, it is impossible for those who have never tasted it to
guess. I finished my education in a school as a pupil teacher, so I can truthfully say, that
after I was seventeen, I maintained myself. You know I was a governess for several years,
but I prefer being a farmer's wife," she concluded with a happy laugh.
"Your lines have fallen in pleasant places," Miss Leighton remarked, with a smile which was
very gracious.
And Mrs. Tiddy agreed.
Then Peggy was asked to give them some music, and she went to the piano willingly. Miss
Leighton was astonished to hear the child could play so well, and expressed herself
delighted, remarking that she had evidently been most carefully taught.
"Soon I am going to learn the organ," Peggy informed the old lady, twisting round on the
piano-stool, "and then, perhaps, when I am quite grown up I shall be able to earn my own
living. How splendid that will be! I think I would rather be a musician than anything else,
because it makes people happy to hear music. Oh! here's Mr. Tiddy!" she cried, catching
the sound of footsteps in the hall; and a minute later the farmer entered the room.
"You've been having some music?" he said, glancing at Peggy on the piano-stool. "Well,
now, won't you sing something, my dear? She has a voice as sweet as a lark's," he
continued, turning to the visitor. "I am sure you would like to hear her sing, wouldn't you?"
"Indeed I should," Miss Leighton replied.
"I don't know any songs," Peggy said doubtfully; "only a few hymns, and little scraps from
anthems which I've heard at church."
"Sing that hymn about 'Light at evening time,' my dear," requested Mr. Tiddy. "I dearly like
to hear you sing that."
Peggy complied immediately, and when her sweet voice ceased there was dead silence for
a minute or so. Surprised, the little girl turned her sightless eyes in the direction of Miss
Leighton, wondering why she did not at least say, "Thank you."
"Don't you like it?" she asked. "It's my favourite hymn, and when I was a very little girl
mother taught me to say the first verse as a prayer. I say it every night now, and I expect
I always shall. I suppose I like it so much because I'm blind. I don't know what light is, but
I know it's very beautiful and wonderful, because Jesus is called 'The Light of the World,'
and people seem to think it's so dreadful to be without it."
"The light our Saviour brought into the world is given to the blind as freely as to others,"
Mrs. Tiddy reminded her gently. "Its home is in the heart, making peace and happiness
and joy." She glanced at Miss Leighton as she spoke and was surprised at the expression
of her face. The old lady was regarding the child with yearning eyes, and her whole
countenance—generally so repellent in its pride—was softened by an emotion which
rendered her incapable of speech.
At that moment Peggy started to her feet, declaring she heard Wolf outside the window—
he was in search of her—and hastened out of the room. A few minutes later, she and her
faithful canine friend ran down the garden path side by side, the dog barking joyously at
having lured her from the house.
"How full of life and high spirits she is!" remarked Mr. Tiddy, as he moved to the window to
watch the pair. "She is looking capital, isn't she? I declare her cheeks have become quite
round and rosy, and she was such a pale little soul not much more than a month ago."
"It is terrible that she should be blind!" Miss Leighton exclaimed, a sort of restrained
vehemence in her tone as she found her voice once more. "Can nothing really be done for
her? Has she had good advice?"
"The best in London, I believe," Mrs. Tiddy answered with a sigh.
"Then money would be no good—" The old lady paused as both her companions shook
their heads. "Because if it was a question of money I would gladly pay any amount for the
child's sake," she proceeded eagerly. "I—I have taken a great fancy to her. I do not know
when I was so much attracted by a child before. I would give a great deal if she could be
made to see."
"Hers is not a case money can touch," Mr. Tiddy responded gravely, "I have been assured
of that by her parents. As long as her life lasts, the little maid will be blind, and she knows
it, but she's contented to wait. Her eyes will see the King in His beauty by-and-by, and
meanwhile His love is lightening her darkness and cheering her way. Did you like that
hymn she sang?"
"Yes," Miss Leighton assented, "but it made me sad. To me, blindness seems the heaviest
affliction that can fall upon any one."
She glanced out of the window, her expression one of mingled affection and pity as her
gaze fell upon the little girl who was now leaning over the garden gate in the attitude of
listening.
"Ah, here comes Barnes to escort me home!" she exclaimed. "I have to thank you for a
very pleasant time," she continued earnestly, looking from one of her companions to the
other. "I am afraid I shall have no opportunity of returning your hospitality now, for I am
leaving Higher Brimley at the end of the week; but surely, Mr. Tiddy, you sometimes bring
your wife to town?"
"She has not been back to London since I married her," Mr. Tiddy replied smilingly, "and
she says she has no desire to go. But I mean for us both to take a holiday in the autumn—
after the corn harvest—and then—"
"And then you will come to London," Miss Leighton interposed quickly, "and do come and
stay with me. Don't say 'No,' but think it over. It would give me so much pleasure to have
you for my guests, and you should do as you pleased in every way. At any rate, promise
you will not visit London without seeing me."
"I readily promise that," Mrs. Tiddy answered, secretly much surprised at the invitation she
and her husband had received. "You are very kind—so many thanks. Won't you stay a little
longer? Barnes can wait for you."
"I think I must go, for I would rather return before dark, and the evening is drawing in.
There is a mist rising from the sea; I dare say it is 'only for heat and pilchards' as you
Cornish folk say, but I am liable to bronchitis and I fear to be out in a fog."
Mr. and Mrs. Tiddy escorted their visitor to the garden gate, where Barnes was waiting for
her, in conversation with Peggy; and five minutes later, mistress and maid were climbing
the hill towards Higher Brimley.
"I shall leave here at the end of the week," Miss Leighton abruptly remarked as they
neared their destination.
"Yes, ma'am," replied Barnes, in her usual quiet tone.
"It is my intention to return to town, but I think I shall break our journey at Plymouth,"
Miss Leighton announced. "I may probably stay there for a day or so."
"Yes, ma'am," Barnes replied again. Not a muscle of her face moved, nor was there any
sign to show the delight she experienced as her mistress made known her plans, though
her heart was palpitating with joy at the thought that she might soon have an opportunity
of seeing her mother and brother.
Miss Leighton was disappointed. She had planned to stop at Plymouth solely on her maid's
account; but of course, she reflected, Barnes could not know that.
"By the way, you have relatives living near Plymouth, have you not?" she asked, after a
brief hesitation.
"Yes, ma'am—my mother and my brother." Barnes regarded her mistress dubiously, then
added: "I shall be glad to see them, if you will allow me a day to myself, for my mother is
very old, and my brother is sorely afflicted—he has no mind, or none to speak of. It will be
a great pleasure to me to go and see them."
"How is it you never mentioned them to me before?" Miss Leighton demanded sharply.
"You are deeply attached to them, are you not?"
"Yes," Barnes admitted, "I am." But she did not explain why their names had never passed
her lips, and her mistress did not ask her again.
CHAPTER X
GOOD-BYES
"HAVE you nearly finished, Barnes?"
The speaker—Miss Leighton—put the question in a querulous tone. She had that moment
entered her bedroom at Higher Brimley, where her maid was engaged in packing her
belongings; and, taking off her bonnet and cloak, she flung them upon the bed with an
irritability of manner which showed she had been put out.
"Yes, ma'am," Barnes answered, as she proceeded to lock the last trunk and securely
fasten its leather straps.
"I have been to Lower Brimley," Miss Leighton announced. "I thought I would call and say
good-bye to the Tiddys this evening, but they have gone to Penzance for the day and
taken the child with them."
There was a distinct note of disappointment in her voice, and her face wore an expression
of mingled annoyance and regret.
"They might have thought that I should call to-day!" she exclaimed, vexedly.
"Do they know you are leaving to-morrow, ma'am?" Barnes questioned, respectfully.
"I told Mrs. Tiddy I intended leaving at the end of this week: probably she imagines that
would be on Saturday—not Friday. I should like to have said good-bye to little Peggy.
Barnes, what I would give if the child's parents would consent to my adopting her!"
"Ma'am!" cried Barnes in great astonishment, rising to her feet—she had been kneeling to
secure the straps of the trunk—and staring at her mistress as though she doubted if she
had heard aright. "Her mother would never permit it!" she declared decisively.
"How do you know?" queried Miss Leighton, with a frown and a cold glance of displeasure.
"Of course I don't know, ma'am," Barnes answered quietly, "and perhaps I have no right to
pass my opinion; but, from what I've heard Miss Peggy say herself, I judge that it's very
unlikely her mother and father would part with her, especially as she's blind. Parents
generally love an afflicted child so much more dearly than those who are better fitted to
face the world!" And Barnes's face softened into tenderness as she spoke.
"But they will have to provide for her future, and my niece's husband is a poor man. If
anything happened to him—if he died, his widow and children would be penniless, and
what would become of Peggy then—helpless and blind? Surely if her parents are so deeply
attached to her, they will consider her interests! I will have nothing to do with Margaret
herself, but she shall not be a loser if she will allow me to adopt Peggy. What do you think
of my plan, Barnes?"
"I don't like it," Barnes responded in a low tone. "No, I don't like it," she repeated, gaining
courage to speak her mind; "the little girl has a happy home, though I suppose it's a poor
one, and she's been accustomed to a great deal of love—"
"And if I did not love her, should I desire to adopt her?" Miss Leighton broke in with
unusual impetuosity.
"Your love is not like that which she's had all her life," Barnes said, refraining from meeting
her mistress's glance. "How can it be, ma'am? You've taken a fancy to the child and you
want her for your own sake, because she's sweet and loveable; but her mother and father
will think of what's best for her—"
The maid's sentence was never finished—and perhaps it was as well, as Miss Leighton's
countenance had darkened with anger—for at that moment Mrs. Ford knocked at the door
with the information that there were visitors downstairs. And on descending to her sitting-
room, the old lady found Mrs. Tiddy and Peggy awaiting her.
"We are so sorry we were not at home when you called, Miss Leighton," said Mrs. Tiddy,
"especially as you are leaving to-morrow—I thought you would not go till Saturday. We
have just returned from Penzance, where we have spent the day."
"I hope you have had a pleasant time," Miss Leighton remarked genially. "But are you not
very tired?"
"I think Peggy is," Mrs. Tiddy replied, "but when we heard you had been to Lower Brimley
in our absence to say good-bye to us, she felt with me that we could not let you go without
a word of farewell, so we decided to come straight on here. We must only stay a few
minutes, though, as my husband is waiting in the dog-cart outside."
"We have had such a lovely day," Peggy informed Miss Leighton. "We had dinner at an
hotel, and we rode to Land's End in a Jersey car; Mr. Tiddy said I must not go home
without having been to Land's End."
"And when do you go home?" Miss Leighton inquired.
"At the end of the month," Peggy answered, "when father is coming to fetch me. It has all
been arranged. Father is going to take a few days' holiday; and I shall be able to show him
the sea, and the village, and the church on the cliff, and all the poultry and the animals on
the farm! Oh! I am so much looking forward to that! But I shall be very sorry when the
time comes to leave Mr. and Mrs. Tiddy and dear old Wolf! I shall never forget my visit to
Cornwall as long as I live! I shall not forget you, either," she went on, taking the old lady's
hand between her own and pressing it. "I don't suppose we shall ever meet again, but I
shall remember you—always. I wish you were not unfriendly with mother! I am sure she
would like to be friendly with you. Don't you think, Aunt Caroline, you might forgive her
now?"
"Did any one tell you to say this to me?" questioned Miss Leighton suspiciously, glancing
from the child to Mrs. Tiddy, who looked somewhat alarmed.
"Oh, no, no! But it seems so dreadful and—and sad that you and mother should not be
friends, for I know you used to be kind to her long ago; and you have been very kind to
me—so different from what I thought you were like!"
"It's my great desire to be always kind to you, Peggy," Miss Leighton said gravely and
impressively. "I wish you to bear that in mind. But you must not meddle between your
mother and me. Little girls should not interfere in matters they do not understand."
Peggy blushed rosy red and her blue eyes filled with tears, but she managed to keep them
back. She felt snubbed and uncomfortable, and was very relieved when Mrs. Tiddy
declared they must go. Miss Leighton rose to escort her visitors to the garden gate, and,
as they were leaving the room, Barnes came downstairs. Peggy recognised the maid's step
immediately, and meet her with extended hands.
"Good-bye, Barnes," she said, adding in a whisper, "I sha'n't forget all you told me about
your poor brother, and I shall remember always to pray for him as I said I would. If you
ever see me in London, you'll be sure to speak to me, will you not?"
"Yes, miss," Barnes responded. She glanced hastily around and saw that her mistress had
followed Mrs. Tiddy out of the front door, then she put her arms around Peggy and kissed
her. "Good-bye, you dear little soul," she said affectionately. "You're going home soon, are
you not, my dear?"
"Yes," Peggy assented happily.
"Ah, you'll be glad to be with your mother and father and brother again, won't you?"
"Indeed I shall," agreed Peggy.
"There's no place like home and the love we get there—remember that, Miss Peggy. It's
better to be rich in love than in money, any day!"
"Of course it is," smiled the little girl. "And I shall be very glad to be at home again,
though Mr. and Mrs. Tiddy have been as kind as kind could be!"
"They're good, kind people, miss; any one can see that, and you've been happy with them,
I know; but—there, I mustn't keep you any longer!" And Barnes kissed Peggy once more
and hurried away.
After that, Peggy hastened to join the others at the garden gate. Mrs. Tiddy had already
taken her place on the front seat of the dog-cart, and Mr. Tiddy was shaking hands with
Miss Leighton and telling her, in his hearty, hospitable way, that she must never pay that
district a visit without coming to Lower Brimley. She assured him that she never would.
"Now then, Peggy. Ready?" he inquired briskly.
The little girl assented, approaching Miss Leighton and holding out her hand. She raised
her face to the old lady's and received a lingering kiss, which she returned rather shyly.
Then, Mr. Tiddy lifted her in his arms and placed her on the back seat of the dog-cart,
bidding her keep a firm hold of the rail of the vehicle and not fall out.
"Good-bye, Aunt Caroline!" cried Peggy brightly, waving her hand, as they started off for
home.
But Miss Leighton made no response. There was a choking sensation in her throat, and she
dared not attempt to speak for fear her voice should betray her emotion. She had a

Neurobiology of Epilepsy From Genes To Networks 1St Edition Elsa Rossignol Full Chapter

Uploaded by

Copyright:

Available Formats

You might also like

Neurobiology of Epilepsy From Genes To Networks 1St Edition Elsa Rossignol Full Chapter

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Neurobiology of Epilepsy From Genes To Networks 1St Edition Elsa Rossignol Full Chapter

Uploaded by

Copyright:

Available Formats

Neurobiology of Epilepsy From Genes

to Networks 1st Edition Elsa Rossignol

Mark Bear, Cambridge, USA.

First edition 2016

Copyright # 2016 Elsevier B.V. All rights reserved

For information on all Elsevier publications

Publisher: Zoe Kruze

Elsa Rossignol, Lionel Carmant, and Jean-Claude Lacaille

1 EPILEPSY AS A SYSTEMS DISEASE

2 INVESTIGATING THE EPILEPTIC NETWORKS: CLINICAL

patients continued having seizures. Unfortunately, no electrode sampled the insula in

validated in the clinical management of epileptic patients, benefiting from increased

2.2 ILLUSTRATIVE CASE

3 INVESTIGATING THE EPILEPTIC NETWORKS:

3.1.2 Combined EEG and fMRI

ballistocardiographic effects, translate into current induction in electrodes. In addi-

in the contralateral temporal and extratemporal regions (Avesani et al., 2014;

3.1.3 Quantified icEEG

3.1.4 Neuroimaging brain networks

3.1.5 EEG/MEG connectivity

(Coben and Mohammad-Rezazadeh, 2015). In addition, according to Elshoff et al.,

3.1.6 fMRI connectivity

3.1.7 Structural connectivity

3.1.8 icEEG connectivity

3.1.8.1 Static network properties

3.1.8.2 Network dynamics: Synchrony

strategy, Burns et al. used a data-driven approach to represent network dynamics

3.1.8.3 Network dynamics: Directionality

3.1.8.4 Network dynamics: The case of ICE

Genetic investigations of the

Progress in Brain Research, Volume 226, ISSN 0079-6123, http://dx.doi.org/10.1016/bs.pbr.2016.04.006

3 MASSIVELY PARALLEL SEQUENCING IN EPILEPSY

that serves as the receptor for the inhibitory neurotransmitter gamma-aminobutyric

Table 1 Novel Epilepsy Gene Discoveries from 2012 to 2014

ALG13 IS, LGS 4 Epi4k Consortium et al. (2013), Michaud

"Do you mean that big man in breeches and leggings?"

"I cannot see," Peggy responded, "because I am blind."

"Blind! How shocking!"

"Every one is surprised to hear I am blind, but it is quite true."

"And have you been blind long?"

"And yet you look happy!" was the wondering exclamation.

"You never take lodgers here?" the stranger inquired hesitatingly.

"A hard, cold voice, wasn't it?" questioned Mr. Tiddy.

"Y-e-s. Was she very old, Mr. Tiddy?"

"Over seventy, I should say."

"No. Listen! The child is singing!"

"Holy Father, cheer our way

"Are you one of a long family?"

"Then you know who she is?"

"Well, that is very generous of her, is it not?"

"Well? What did he say?"

"I will have no more to do with her," she thought.

"No, my dear, I do not," he agreed.

"Indeed I do; so we have that much in common, at any rate."

"You do not go on the cliffs alone?" Miss Leighton interposed.

"Yes, Miss Leighton," Peggy answered, smiling.

MISS LEIGHTON'S maid—Ellen Barnes—was a plain, sad-faced, middle-aged woman who

"Oh, yes, I am sure she does!" Peggy agreed earnestly.