Association between pre-stimulus microstates and

evoked activity in an auditory gating paradigm in

22q11.2 deletion syndrome. A high-density EEG study.

Master in Neuroscience

Alexandra Souchkova

Jury Members:
Prof. Thomas Koenig
Prof. Christoph Michel
Dr. Tonia Rihs
Dr. Miralena Tomescu
 Table of Contents

Abstract ............................................................................................. 4
I. Introduction .................................................................................. 5
1.1 22q11.2 Deletion Syndrome ........................................................ 5
1.1.1 Nomenclature, phenotypes, genetics .................................... 5
1.1.2 Clinical profile ..................................................................... 6
1.1.3 Cognitive profile .................................................................. 6
1.1.4 Psychiatric profile and susceptibility to schizophrenia ....... 7
1.2 EEG Auditory Evoked Response Potential.................................. 8
1.2.1 Basic anatomical and functional properties of audition ...... 8
1.2.2 EEG and event-related potential.......................................... 9
1.2.3 Auditory ERP components ................................................. 10
1.2.4 Auditory sensory gating ..................................................... 10
1.2.5 Auditory sensory gating in schizophrenia and 22q11.2 DS
..................................................................................................... 11
1.3 EEG microstate analysis and neural networks ........................... 13
1.3.1 Principles of neural networks ............................................ 13
1.3.2 EEG microstate analysis at rest ......................................... 14
II. Materials and methods ............................................................. 17
2.1 Participants ................................................................................. 17
2.2 Task ............................................................................................ 18
2.3 EEG recording and processing................................................... 19
2.4 ERP Analysis ............................................................................. 19
2.5 Microstate analysis..................................................................... 21
2.6 Correlations with symptoms ...................................................... 22
III. Results ...................................................................................... 22
3.1 Auditory evoked response results .............................................. 22
3.2 Pre-stimulus microstate analysis results .................................... 24
3.3 Microstate and evoked activity associations .............................. 27
3.4 Correlations with clinical symptoms ......................................... 27
IV. Discussion ................................................................................. 28
4.1 Central N1 in 22q11.2 DS .......................................................... 28
4.2 Microstate B, C and D dynamics in 22q11.2 DS ....................... 29
4.2.1 Class B increase in 22q11.2 DS ......................................... 29
4.2.2 Class D decrease in 22q11.2 DS and schizophrenia ......... 30

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 4.2.3 Class C dynamics and symptoms in 22q11.2 DS ............... 31
4.3 Microstate class A and N1 latency in 22q11.2 DS .................... 33
4.4 Limitations and future directions ............................................... 33
4.5 Conclusion ................................................................................. 34
V. Bibliography .............................................................................. 35
VI. Appendix .................................................................................. 42
5.1 Correlations with clinical symptoms ......................................... 42

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Acknowledgements

I would like to thank Professor Michel for the opportunity to do my

Masters research in the Functional Brain Mapping Lab. I would also
like to express my appreciation for the guidance, teachings, patience
and expertise of Dr. Tonia Rihs and Dr. Miralena Tomescu
throughout this project. A special thank you to Dr. Anna Custo for
helping me in the Matlab analysis of auditory gating. Notably, this
work could not have been possible without the love of my mother and
father, the faith of my grandparents, the support of wonderful friends
in the USA and in Europe and the motivation of Scott Gregg. Finally,
I would like to recognize the 22q11.2 deletion syndrome patients and
their families who committed to participating in this research, as it has
been entirely humbling to witness their courage.

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Abstract

Previous studies have used EEG as a non-invasive method with high temporal
resolution to examine biomedical markers of schizophrenia onset in 22q11.2
deletion syndrome (22q11.2 DS), a genetic disorder distinguished by a severe
vulnerability to schizophrenia. Research investigating the auditory event-
related potential (AERP) in these groups has reported an increased amplitude
of the central N1 component in 22q11.2 DS and a decreased amplitude of this
component in schizophrenia in an auditory gating paradigm, suggesting
compromised auditory processing. Additionally, research on the global
functional state of the brain in 22q11.2 DS and schizophrenia has used
microstate analysis to show an increased presence of microstate class C and
a decreased presence of class D at rest in both groupsdemonstrating
abnormal saliency and attentional processing, respectively (Andreou et al.,
2014; Koenig et al., 1999; Lehmann et al., 2005; Tomescu et al., 2014;
Tomescu et al., 2015). The present study uses high density EEG to investigate
the relationship of microstate dynamics to the central N1 AERP component
in a paired click auditory task as participants (15 22q11.2 DS patients and 19
healthy age-matched controls) watch a muted cartoon. The findings observe
an increased central N1 GFP amplitude, consistent with previous research
(Rihs et al., 2013), as well as a shortened central N1 latency in patients, which
could be due to impaired auditory processing specific to 22q11.2 DS.
Microstate results show an increase in class B, thought to be associated with
visual processing, thus we postulate this to relate to increased visual
stimulation in 22q11.2 DS in this eyes open condition. We also observe
decreases in classes C and D in 22q11.2 DS; the decrease in class C is
inconsistent with previous findings on resting state in the group (Tomescu et
al., 2015) and may suggest the saliency network to be particularly sensitive
to eyes open vs. eyes closed conditions, while the decrease in class D appears
to be stable across conditions and comparable to research on schizophrenia,
implying a robust higher order processing deficit in both groups.
Furthermore, we report an association of microstate class A and central N1
latency in 22q11.2 DS, and an inverse association of these with clinical
symptoms, which relates auditory processing to psychiatric disorder.
Prominently, these results express a decreased class D in 22q11.2 DS as a
reliable EEG signature of the group, similar to research on schizophrenia,
central N1 AERP hyperactivity as a feature of 22q11.2 DS, and microstate C
dynamics that could be context-dependent.

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I. Introduction

22q11.2 DS and Schizophrenia are severe disorders with shared

elements. 22q11.2 DS is a genetic disorder that puts patients at high
risk of developing schizophrenia (Karayiorgou et al., 2010).While the
genetic microdeletion responsible for 22q11.2 DS is apparent, the root
cause of schizophrenia is unknown (McGuffin et al., 1995). In the
following exploration, I will discuss the characteristics of 22q11.2
DS, its relationship to schizophrenia, previous research on sensory
and higher order processing deficits in these groups, and EEG as a
method of studying these deficits in evoked response and pre-stimulus
conditions in prospect of researching schizophrenia etiology.

1.1 22q11.2 Deletion Syndrome

1.1.1 Nomenclature, phenotypes, genetics

22q11.2 deletion syndrome is a genetic disorder that has an estimated

prevalence of one in 4000 births (Bassett et al., 2011) and has several
names including DiGeorge syndrome and velocardiofacial syndrome.
DiGeorge Syndrome was coined in 1966 based on Dr. DiGeorge
reporting that instances of infants born without a thymus could serve
as human homologues for animal research on the dichotomy of the
lymphoid system (Greenberg, 1993). The term velocardiofacial is
descriptive of the syndromes phenotypes: velo referring to
structural and functional palatal abnormalities, cardio denoting
cardiac effects, and facial due to unique facial features that are
characteristic of patients, such as hooded upper eyelids (41%)and
other ocular impairments including ptosis (9%) and distichiasis
(3%)and small and/or protuberant ears, as well as squared helices
(McDonald-McGinn et al., 1993). Other phenotypes of the syndrome
include scoliosis (45%), short stature (20%), strabismus (15%)
(Bassett et al., 2011).

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22q11.2 deletion syndrome describes the genetic origin of the
syndrome: a microdeletion or translocation of a section of
chromosome 22, of up to 3 megabases (Mb) in size, containing
approximately 60 known genes (Karayiorgou et al., 2010). q
describes that this region is on the long arm of the chromosome 22,
and 11.2 depicts the exact location of the deletion (Scambler, 2000).
This chromosomal region is highly vulnerable to mutation which is
perhaps why most 22q11.2 DS cases are due to a de novo mutation
(90-95%), while only about 10% of cases are inherited (Demily et al.,
2015). Moreover, given the variability of deleted genes and of the
homologous undeleted region between cases, the disorder manifests
in a specific manner in each afflicted individual (Bassett et al., 2011).
Because of this variety in manifestation of the syndrome the condition
has been studied by clinicians of different specialties, and has in turn
been described using diverse titles such as conotruncal anomaly face,
autosomal dominant, opitz G, Sedlackova and Cayler cardiofacial
syndrome (Bassett et al., 2011; McDonald-McGinn et al., 1993). For
the remainder of this report, I will refer to the condition as 22q11.2
deletion syndrome as it is now commonly discussed using the
chromosomal etiology (Bassett et al., 2011).

1.1.2 Clinical profile

The assortment of clinical symptoms expressed in 22q11.2 DS differs

per patient, yet there are several indications of the condition that are
highly prevalent. Common clinical symptoms include cardiovascular
malformation, immune deficiency, thymic abnormality,
hypocalcemia and hypoparathyroidism (Swillen et al., 2000).
Prominently, congenital heart defect was observed to be present in
74% of individuals in a study of 250 patients with 22q11.2 DS. It was
also shown to be the leading cause of mortality in this diagnosis
(>90% of deaths) (McDonald-McGinn et al., 2001).

1.1.3 Cognitive profile

While indicators and severity of impairment also vary between

22q11.2 DS patients, neuropsychological tests have revealed a
generalizable cognitive profile of the cohort. Collectively, the group

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tends to have IQ scores that are below average or edging on the
threshold (Swillen et al., 2000). This results in an overwhelming
majority90%of the patients having learning difficulties
(Lindsay, 2001), and in turn being limited in life and career
opportunities (Bassett et al., 2011). In addition, people with 22q11.2
DS have a particularly difficult time with perceiving and constructing
speech and language both due to their cognitive and physical
abnormalities (D'Antonio et al., 2001).

1.1.4 Psychiatric profile and susceptibility to schizophrenia

22q11.2 DS patients exhibit psychiatric comorbidities in addition to

cognitive deficits. Behavioral observations of 22q11.2 DS report both
disinhibited and impulsive temperament as well as shyness and
withdrawal (Swillen et al., 1999). In childhood, ADHD and autism
spectrum symptoms are most commonly apparent in the group
(Bassett et al., 2011). ADHD is observed in over a third of 22q11.2
DS cases, and is inherently linked to cognitive disabilities (Schneider
et al., 2014), while autism-like behavior appears in about a quarter of
patients and is thought to be due to hypocalcemia (Muldoon et al.,
2015). Anxiety and depressive disorders are also commonly present
in childhood, adolescence and adulthood (Bassett et al., 2011). Given
the plethora of findings on psychiatric comorbidities in 22q11.2 DS
Schneider and colleagues (2014) conducted a meta-analysis of 15
studies on 1,402 patients (6-68 years) and showed that ADHD was
observed in 37% of children, while anxiety disorder was reported in
35% of this age group. Autism spectrum disorders were present in
26% of adolescents (13-17 years). Mood disorder increased with age,
and was shown to have a prevalence of 18% in emerging adults (18-
25 years) (Schneider et al., 2014).

As many psychiatric challenges surface in childhood and

adolescence, 60% of 22q11.2 DS patients are diagnosed with
psychiatric disorders in adulthood (Bassett et al., 2011; McDonald-
McGinn et al., 2001). Interestingly, 22q11.2 DS patients are
extremely susceptible to schizophrenia, having a 30% chance of
schizophrenia onset as early as in young adulthood (Murphy et al.,
1999). The onset of schizophrenia has been shown to indeed be

7
significantly earlier in 22q11.2 DS patients than in other
schizophrenia cases (Debbane et al., 2006). The aforementioned
analysis by Schneider et al. (2014) reported the prevalence of
schizophrenia to be 41% by full adulthood (36 years). This strong
relationship could be due to the shared genetic origin of schizophrenia
and 22q11.2 DS (Coon et al., 1994).

22q11.2 DS can serve as a reliable model for researching biomarkers

of schizophrenia etiology based on the high risk of development
(Debbane et al., 2006), genetic link to (Coon et al., 1994), and early
onset of the disease in 22q11.2 DS compared to people with
schizophrenia in the general population (Debbane et al., 2005).
Notably, the lack of clinical differences between the schizophrenia
phenotype in the general population and that of schizophrenia in
22q11.2 DS also supports investigation of biomarkers of
schizophrenia in the group (Karayiorgou et al., 2010). In addition,
22q11.2 DS is the third highest risk factor for development of
schizophrenia, next to having a monozygotic twin with schizophrenia
(50% risk), and having two schizophrenic parents (46% risk), making
22q11.2 DS patients a vital sample for research (McGuffin et al.,
1995). In the investigation of biomarkers, the EEG auditory evoked
response has previously been examined as a possible indicator which
could foreshadow schizophrenia onset in the group (Rihs et al., 2013).

1.2 EEG Auditory Evoked Response Potential

1.2.1 Basic anatomical and functional properties of audition

Human audition is possible due to a system of dynamic processes

which are formally organized into the central auditory system and the
peripheral auditory system. The peripheral auditory system includes
hair cells of the cochleaa fluid filled structure inside the earthat
transform the pressure of sound waves into neural signal which then
innervates a bundle of fibers known as the auditory nerve, or cranial
nerve VIII (Purves, 2004). Next, the auditory nerve projects to the
central auditory system where several nuclei in the brainstem synapse
before reaching the inferior colliculi. From the inferior colliculi, the
information travels to the medial geniculate complex of the thalamus,

8
which relays the information to the primary auditory cortex (Purves,
2004). The auditory cortex makes connections with a wide range of
other cortical areas for higher order processing of information which
is imperative for the interpretation of sound in the context of the
environment (Purves, 2004). Given that audition in the human brain
involves a sophisticated network, a dysfunction of any of the parts
involved can result in effects that seriously impact daily functioning.
The present study focuses on higher order processing of sound at the
cortical level.

1.2.2 EEG and event-related potential

EEG is a non-invasive brain imaging method that employs electrodes

placed at the surface of the scalp to measure the electrical potential
field created by the post-synaptic potential of synchronous pyramidal
neurons activity (Michel et al., 2009). Pyramidal neurons are aligned
parallel to one another and perpendicular to the circumvoluted
cortical surface (Michel et al., 2009). In turn, directionality of the
electric dipole field created by the post-synaptic potential determines
the different potential distribution recorded by each electrode (Michel
& Murray, 2012). Electrodes potentials must be subtracted from a
chosen reference electrode, thus the value of the reference is 0 V by
definition (Michel et al., 2009). Consequently, potential values
recorded at each electrode are reference-dependent, i.e. susceptible to
change based on the chosen reference (Michel et al., 2009).

Two reference independent measures that address this issue are the
Global Field Power and Global Map Dissimilarity (Michel et al.,
2009). The GFP is the standard deviation of the average referenced
potentials at each electrode and represents the strength of the electric
field over the scalp at a given moment in time, independent of the
reference used for the recording (Michel et al., 2009). The maximum
of the GFP curve indicates the strongest field strength and highest
signal to noise ratio within the time sequence (Michel et al., 2009).
The GMD compares the topographical differences between potential
maps and thus describes the stability of underlying neuronal activity
in a time series (Murray et al., 2008).

9
The high temporal resolution of EEG allows for researchers to
examine the changes in intensity and distribution in the potential field
in response to a presented stimulus at the millisecond level (Michel et
al., 1999). A stimulus can generate a neural response and thus, a
different potential field will be measured on the scalp. This event-
related potential (ERP) is time locked to the stimulus presentation; in
turn, averaging many repetitions of the ERP will improve the signal
to noise ratio (Michel et al., 1999).

1.2.3 Auditory ERP components

Auditory evoked responses, or changes in the electrical potential field

at the scalp in response to an auditory stimulus, are composed of
components that occur at different stages of auditory processing
(Naatanen & Picton, 1987). I will namely discuss the P50 and the N1
components of the auditory ERP (AERP). The P50 is an early
auditory response occurring at about 50 milliseconds post stimulus
presentation (Liegeois-Chauvel et al., 1994). The P50 spatial
allocation of the potential field results in a frontal positivity and its
neuronal generators are thought to be widely distributed in the
primary auditory cortex (Liegeois-Chauvel et al., 1994). The N1
occurs between 80 and 120 ms post stimulus presentation, and
initially has a spatial topography of central negativity, known as the
central N1. It subsequently evolves into the lateral N1 in which the
negativity spreads horizontally to either sides of the scalp (Naatanen
& Picton, 1987). The N1 is attributed to higher order auditory
processing and involves primary and secondary auditory cortices, and
planum temporale (Naatanen & Picton, 1987). Though the stability
and reliability of these components in healthy subjects has been
established through replication, the cerebral generators of the AERP
remain controversial, especially those of later components which
seemingly recruit a broader neuronal network (Liegeois-Chauvel et
al., 1994).

1.2.4 Auditory sensory gating

Human sensory processing functions in a way that allows for

efficiency in an environment of sensory overload; the brain

10
orchestrates this by subconsciously muting redundant stimuli, which
allows for focus to be directed to that which is contextually relevant.
Auditory sensory gating describes the decrease in the response of the
auditory system when presented with successive presentation of an
auditory stimulus (Fruhstorfer et al., 1970). This is demonstrated by
a classical auditory sensory gating paradigm where a series of paired
click sounds are presented to participants (Fruhstorfer et al., 1970). In
healthy people the P50, N100, P200 AERP components are observed
to be stronger for the first click than for the second click (Fruhstorfer
et al., 1970).

Studies on auditory gating have shown that P50 gating is associated

with a pre-attentional inhibitory filter (Freedman et al., 1987; Jerger
et al., 1992; White & Yee, 1997), that could protect higher order
functioning (Freedman et al., 1991; Jerger et al., 1992; Wan et al.,
2008), while N100or N1gating is thought to be related to filter
mechanisms involved in triggering of attention (Lijffijt et al., 2009).
A study of sensory gating on 56 healthy adults P50 gating showed
that P50 gating is related to less commission errors in a delayed
memory task, reflecting the ability to inhibit a response generated
before a stimulus is fully analyzed (Lijffijt et al., 2009). The study
also exemplified that stronger N1 gating was shown to be related to a
higher sensitivity for discriminating between a target and an off-target
stimulus in the taska sensitivity that is affected by attentional and
perceptional processes (Lijffijt et al., 2009). In comparison to healthy
subjects, P50 gating (Cullum et al., 1993; Freedman et al., 1987;
Turetsky et al., 2007) and N1 gating has been shown to be impaired
in the presence of mental disorder (Ford et al., 2001; Foxe et al., 2011;
O'Donnell et al., 2004; Ogura et al., 1991; Potts et al., 1998; Salisbury
et al., 2010) suggesting a descriptive relationship to mental
dysfunction.

1.2.5 Auditory sensory gating in schizophrenia and 22q11.2 DS

Auditory processing has been shown to be deficient in schizophrenia

and the auditory ERP has been recognized as a reliable biomarker for
the disease (Calkins et al., 2007; Greenwood et al., 2011; Turetsky et
al., 2007). In a study using the double-click auditory gating paradigm

11
Turetsky and colleagues (2007) showed the impairment of a healthy
suppression of the P50 and N1 responses to the second click in
schizophrenia patients. Rihs and colleagues (2013) employed the
same paradigm to study auditory gating in 22q11.2 DS and found this
healthy suppression to be preserved in patients. Additionally, an
increased amplitude of the central N1 response to the first click was
observed to be a feature of the 22q11.2 DS group (Rihs et al., 2013).
In contrast to P50 gating, which seems to be impaired in
schizophrenia and not in 22q11.2 DS, the central N1 component has
been shown to be abnormal in both groups (Ford et al., 2001; Foxe et
al., 2011; O'Donnell et al., 2004; Ogura et al., 1991; Potts et al., 1998;
Rihs et al., 2013; Salisbury et al., 2010). Still, though the N1 may
deviate from healthy subjects in schizophrenia and 22q11.2 DS
patients, numerous studies on schizophrenia have shown a reduction
in amplitude of the central N1 AERP component in response to the
first click (Ford et al., 2001; O'Donnell et al., 2004; Ogura et al., 1991;
Potts et al., 1998; Salisbury et al., 2010) and not in 22q11.2 DS (Rihs
et al., 2013). Findings on the increased central N1 amplitude in
response to the first click in 22q11.2 DS patients (Rihs et al., 2013)
are illustrated by Figure.1 below.

12
 Figure.1 Rihs et al. (2013)
demonstrated an increase in the
amplitude of the central N1 AERP
component in response to the first
click in an auditory gating
paradigm 22q11.2 DS patients
compared to healthy controls.

1.3 EEG microstate analysis and neural networks

1.3.1 Principles of neural networks

The human brain contains widely distributed neural networks of

interconnected cortical areas which allow for parallel information
processing in a rapidly changing and highly stimulating environment
(Bressler, 1995). The synergy of these networks allows for healthy
mental performance, while the converse could be associated with
neuropsychiatric illness (van den Heuvel & Hulshoff Pol, 2010).
Studies have shown that contrary to supposition, when one is not
engaged in any specific activity neural networks are highly active;
these resting state networks have been studied using fMRI, where
organized hemodynamic response to neural functioning in the

13
absence of external stimuli has been associated with visual, auditory,
executive, and memory functions, among others (Damoiseaux et al.,
2006; Snyder & Raichle, 2012). EEG offers high temporal resolution
at the millisecond level and measures neuronal signaling directly
(Michel et al., 2009), making it an advantageous method of analysis
of such networks compared to fMRI which has a lower temporal
resolution and measures indirect metabolic signals. I will explore this
method further in the following section.

1.3.2 EEG microstate analysis at rest

Research on resting statei.e. a task-negative condition when

participants are not being met with any sensory or cognitive
demandshas identified recurring signature topographic
representations of the scalp potential field that are quasi-stable in
time, lasting between 80 and 120 ms (Lehmann & Skrandies, 1980;
Lehmann et al., 1998). Koenig and colleagues (1999) defined 4
microstates in which the topographies and temporal parameters of
each state were consistent across a wide range of EEG recordings and
explained 80% of the variance in the data. Britz et al. (2010) followed
to demonstrate that microstates previously labelled A, B, C, and D
correspond to fMRI resting state networks associated with auditory,
visual, saliency and attention processing, respectively.

The dysfunction of said networks can be described in terms of

different temporal characteristics, such as the activation of a certain
network for an abnormal duration. Due to this, four temporal
parameters are commonly used to measure microstate dynamics:
frequency of occurrence, mean duration, time coverage, and global
explained variance (Khanna et al., 2015; Lehmann et al., 1987;
Lehmann et al., 1998). The frequency of occurrence is the average
number of times per second a microstate topography is dominant, and
can be interpreted as the likelihood of the activation of specific
neuronal assemblies (Khanna et al., 2015; Lehmann et al., 1987). The
mean duration is the average time (in milliseconds) that a microstate
remains stable within the EEG recording, and thus can be interpreted
as the average interval of the activation of specific neuronal
assemblies (Khanna et al., 2015). The time coverage describes the

14
percentage of the entire EEG recording that corresponds with a
microstate topography (Lehmann et al., 1987). Lastly, the global
explained variance is the sum of the explained variances of each
microstate weighed by the GFP (Brodbeck et al., 2012). Both latter
measures can be interpreted as the total time neural generators
specific to a microstate are active in comparison to those of other
microstates (Khanna et al., 2015).

1.3.3 Microstates at rest in schizophrenia and 22q11.2 DS

An advantage of microstate analysis is that it exemplifies global

functional states of the brain in health and illness (Khanna et al.,
2015). Previous studies have shown similar deviant resting state
patterns in both 22q11.2 DS and schizophreniaspecifically an
increase in the presence of class C and a decrease in the presence of
class D (Andreou et al., 2014; Koenig et al., 1999; Lehmann et al.,
2005; Tomescu et al., 2014; Tomescu et al., 2015). Microstate class
D is associated with higher order cognitive processing involving
reorientation of attention (Britz et al., 2010); the decrease of this
microstate at rest in 22q11.2 DS and schizophrenia could be related
to impaired cognitive abilities that are characteristic of patients
(Karayiorgou et al., 2010; Lewandowski et al., 2007). Studies have
suggested that the salience resting state network, associated with class
C, is involved in discriminating and integrating internal and external
stimuli as well as recruiting sensory information processing networks
(Britz et al., 2010; Tomescu et al., 2014). This research proposes that
a dysfunctional saliency resting state network could decrease the
activation of sensory information processing networks in 22q11.2 DS
and schizophrenia, and may results in aberrant top-down processing
as a whole (Tomescu et al., 2015). Findings by Tomescu et al. (2015)
on the increase of class C and decrease of class D in 22q11.2 DS and
schizophrenia are illustrated by Figure.2 below.

15
 * **
Mean duration (ms)

* *

Figure.2 Tomescu et al. (2015) demonstrated a significant increase of mean duration of

microstate class C and decrease of microstate class D in 22q11.2 DS and schizophrenic
patients. (*<0.05 **<0.001)

1.4 Aims of the present study

Aberrant microstate activity during rest in 22q11.2 DS has been

shown to be similar to microstate activity at rest in schizophrenia,
making this a possible risk marker for the development of the disorder
(Tomescu et al., 2015). AERP components have also been shown to
deviate from healthy subjects in both 22q11.2 DS and in
schizophrenia (Calkins et al., 2007; Greenwood et al., 2011; Rihs et
al., 2013; Turetsky et al., 2007). Thus, the present study aims to
answer how abnormal microstate dynamics in 22q11.2 DS relate to
the irregular central N1 component observed in the group in response
to the first click a double-click auditory gating paradigm (Rihs et al.,
2013). Additionally, we would like to know how these EEG
parameters relate to psychiatric symptoms in the patients. To do this,
the present study analyzes microstate dynamics between auditory
stimulus presentation in the auditory gating paradigm, as well as the
AERP, measures association between these, and attempts to align
them to psychiatric symptoms.

16
II. Materials and methods

2.1 Participants

The data analyzed for this study were derived from the NCCR-
Synapsy study on 22q11.2 deletion syndrome. Participants for the
EEG recording were recruited through ads in patient association
newsletters. 31 controls (11 female; 15.129 2.3486; mean S.D.) 28
patients (12 female; 15.75 2.7028; mean S.D.) with no recorded
auditory deficit were selected for the study. There was significant
overlap with subjects analyzed in the Tomescu et al. (2014) study on
resting state and with both subjects and data analyzed in the Rihs et
al. (2013) research on sensory gating. The age selection criterion was
12 to 19 years old, to remain consistent with Tomescu et al. (2014)
research on resting state microstates in adolescents.

All participants were tested on a full Wechsler Intelligence scale for

children III-R (WISC-III-R) or the Wechsler Adult Intelligence
Scale III (WIAIS-III) for those 17 years of age or older. There were
significant differences between the two analyzed groups on the full-
scale IQ (FSIQ) as confirmed by an independent sample t-test; FSIQ:
[22q11.2 DS: 72.24 11.12; controls: 112.73 17.53; mean S.D.,
t(df=36) = -12.28, p=0.000].

Parents/caregiver of the 22q11.2 DS patient, or the patient if he or she

was 18 or older, underwent a structured clinical interview for DSM-
IV axis I disorders (SCID) and were excluded from the analysis if
they had record of schizophrenia, schizophreniform or
schizoaffective disorders at the time of the evaluation.

Concatenation of four separate trials of the auditory task, marking of

artifacts, independent component analysis, averaging, extracting of
the pre-stimulus epochs and individual k-means clustering was
performed on all 59 subjects. This process is illustrated by Figure.5
in the ERP Analysis section. 15 subjects were then excluded from the
group k-means clustering analysis due to excessive eye blinks,
movement and noise; thus 23 controls (9 female, 14.86 2.398; mean
S.D.) and 21 patients (8 female, 15.66 2.869; mean S.D.) were

17
included in the group k-means clustering, and the spatial correlation
with the topographies resulting from the group k-means clustering.
An independent sample t-test was conducted on the ages of 22q11.2
DS patients and controls and revealed no significant age differences
between the two groups [t(df=39) = 2.02, p=0.502]. This process is
illustrated by Figure.3 in the ERP Analysis section.

19 controls (7 female, 15.1 2.306; mean S.D.) and 15 (7 female,

15.87 2.89; mean S.D.) were used for the correlation of N1
amplitude and microstate temporal parameters. 10 participants were
excluded due noise in the ERP signal or no clear central N1 GFP peak.

A subset of these participants was included in examination of P50

sensory gating due to clear GFP peaks of the first and second P50 only
present in part of the cases, 11 controls (4 female, 14.72 1.95; mean
S.D.) and 13 patients (5 female, 15.3 2.926; mean S.D.).
Additionally, 23 subjects where clear peaks were identified on the Cz
electrode after average referencing were selected for an additional
measure of P50 gating, 11 controls (2 females, 14.54 2.11; mean
S.D.) and 12 patients (5 females, 15.58 2.874; mean S.D.).

2.2 Task

During the EEG recording, participants sat upright in a Faraday cage

and watched a muted cartoon. Auditory stimuli were delivered
through insert earphones (Etymotic Research, Elk Grove Village, IL,
USA). Participants heard 120 binaurally presented paired-click tones
(1.5 ms, 86-dB SPL) with 500 ms between the two tones, and between
10 to 12 s between the pairs. 4 consecutive trials of the session were
conducted with short pauses in between each trial. An illustrative
representation of the task and analysis is shown by Figure.3 below.

18
Task:
Condition: watching silent cartoon

12 seconds between click pairs 12 seconds between click pairs

500 ms
between clicks

Analysis:

8000 ms 1100 ms

Microstate dynamics observed: ERP (a) observed: ERP (b) observed:

Class A 1st P50 2nd P50
Class B 1st central N1 2nd central N1
Class C 1st lateral N1 2nd lateral N1
Class D

Figure.3 Participants watched a silent cartoon during the presentation of the click pairs.
Microstate dynamics of the periods for the 8000 ms preceding the first click were
analyzed. The AERP to the click pairs were analyzed from 200 ms before the first click
to 900 ms (1100ms total) after the first click, to cover the evoked response for the whole
interval of stimulus presentation.

2.3 EEG recording and processing

EEG data was acquired through a 256-channel hydrocel cap

(Electrical Geodesics Inc., Eugene, OR, USA), with a sampling rate
of 1000 Hz. Offline, four separate trials of the task were concatenated.
Next, muscle artifacts and bad electrodes were manually marked by
the author of this manuscript. The electrocardiogram and eye
movements were removed using infomax independent component
analysis implemented in Matlab by Frederic Grouiller (Jung et al.,
2000; Onton et al., 2006). The script filtered data between 1 and 40
Hz and removed cheek and neck electrodes from the signal, thereby
reducing the data from 256 to 204 channels. Cartool Software
(fbmlab.com/cartool-software) was used for data processing
described in the following analyses.

2.4 ERP Analysis

The evoked response to the click sounds was analyzed to:

Distinguish the GFP local maximum of the central N1

component.
Measure sensory gating at the level of the P50 component.
Identify the latencies of the central N1, first and second P50
components.

19
For these analyses the data were averaged from -200 ms before to 900
ms after the first click. Visual inspection of epochs was performed by
the author of this manuscript to exclude periods contaminated by
excess noise. No baseline correction was applied. Bad channels were
removed and interpolated i.e. replaced by estimations that were
computed using the bad channels geometrical neighbors (Michel et
al., 2009). The data were re-referenced to a common average
reference (Michel et al., 2009). GFP, GDM and manual inspection of
topographies were used to identify the central N1, lateral N1 and P50
components.

To investigate control and patient group differences in signal

amplitude, an exploratory analysis of all time points and all electrodes
was performed using a randomization test in Cartool. A significance
level of p<0.05 was fixed and a temporal criterion of at least 10 ms of
significance was added to ensure that the significant level was
maintained for at least 10 ms.

For auditory gating analysis, ERP data were filtered from 10 to 40 Hz,
as identification of the P50 can be hampered by lower frequencies in
the signal (Jerger et al., 1992; Olincy et al., 2010). Analysis of GFP
and the Cz electrode amplitude reduction in response to the second
click at the P50 was performed by subtracting the absolute value of
the difference of the local maximum and minimum amplitudes of the
second P50, from the absolute value of the difference of the local
maximum and minimum of the first P50. This method of analysis is
in line with classical ERP analyses and was performed to relate our
results to the literature in the P50 gating field (Lijffijt et al., 2009;
Rihs et al., 2013; Smith et al., 1994). For this procedure, intervals in
which the P50 GFP and Cz peaks occurred were identified by visual
inspection by the author of this manuscript and read into a Matlab
script, which performed the calculations. Two-tailed independent
sample t-tests were performed on the two groups for both measures,
using Statistica software (statsoft.com/Products/STATISTICA).
Figure.4 below illustrates the analysis pipeline.

20
 ICA:
Grand Average
Removal of Submission to
cardiac/eye artifacts AERP EEG
Trial 1 Grand Average
& filter 1-40 Hz
N=31 N=28 Visual Inspection: Averaging: N=19 N=15 Central N1 GFP
Trial 2 Artifact marking Selection of epochs Visual inspection: Maximum
Concatenated Artifact Cleaned Individual Averaged
& identification of Interpolation: AERP component
EEG EEG AERP EEG identification
Trial 3 bad channels Removal of Central N1 GFP
bad channels Latency
P50 Gating
Trial 4
Pre-stimulus epochs isolation: Matlab Script P50 Gating Metrics
Selection of epochs corresponding to AERP analysis only

Individual Pre-stimulus
Epochs EEG

Downsampling to 125 Hz
Interpolation Microstate
Artifact Marking N=19 Central N1
N=15 Symptom
Processed Individual Pre-stimulus Association Analysis
Epochs EEG

Submission to Individual
K-means Clustering

N - Controls N=23 Individual K-means

N - 22q11.2 DS N=21 topography solutions

Submission to Group K-means

Group K-means
topography solutions

Spatial Correlation

Microstate Temporal
Parameters

Figure.4 A schematic representation of the analysis pipeline for the investigation

of the AERP, sensory gating, and microstate dynamics.

Latencies of the central N1, and first and second P50 were calculated
by subtracting the time point of the stimulus onset from time point of
the components GFP maximum. Two-tailed independent sample t-
tests were performed on the two groups for these three measures using
Statistica software.

2.5 Microstate analysis

To select periods for microstate analysis, data were selected -8000 ms

before the presentation of the first click using only epochs
corresponding with those used for the ERP analysis. This selection of
epochs was done to be able to investigate the association between
microstate activity and the AERP. Data were interpolated, down-
sampled to 125 Hz, and recomputed to an average reference. Artifact
periods were marked by the author of this manuscript. Data were then
submitted to individual k-means clustering analysis. The k-means
clustering used the GFP to identify the most representative
topographies of the EEG which were interpreted as the dominant
microstates for that individual. The individual microstate topography
solutions ranged from 1 to 10 per subject. These topographies were

21
applied to the group k-means clustering analysis. The results of the
group k-means clustering proposed the optimum criteria (based on an
average of multiple algorithms including cross-validation) of 4 maps
for each group. To examine the mean duration, time coverage,
frequency of occurrence, and global explained variance of each
microstate for per individual, a spatial correlation between the groups
dominant microstate topographies and the topographies
corresponding to the individuals EEG at each time point was
performed. Figure.5 demonstrates this process.

2.6 Correlations with symptoms

Interviews with parents/caregiver of the participant, or the participant

if he or she was 18 years or older, were administered the day of the
EEG recording (1 day) and symptom severity was recorded using
several scales. The Positive And Negative Symptom Scale (PANSS,
Kay et al., 1987) is a 30 item scale composed of a positive, negative
and a general psychopathology symptoms scale. All raw scores were
converted to t-scores using the PANSS manual. The Brief Psychiatric
Rating Scale (BPRS; Overall and Gorham, 1962) is a 24-item scale
where the severity of each symptom is rated from 1 to 7. Scores for
each symptom, as well as the total score, were reported. Microstate
temporal parameters and central N1 GFP maximum, central N1
latency were correlated with symptom severity scores using Statistica
software.

III. Results

3.1 Auditory evoked response results

Both controls and 22q11.2 DS patients showed the known reduction

of the second P50 amplitude in the evoked response to the second
click as opposed to the first click. The randomization test comparing
all electrodes at all time points at a fixed significance level of p<0.05
where the significance level had to be maintained for at least 10 ms

22
 showed a significant difference between groups for the first central
N1 component. This is illustrated by Figure.5 below.

A.
V - 22q11.2 DS Figure.5 (A) The grand average
- Controls
AERP for the first click (500 ms)
2 in the two groups (22q11.2 DS
N=15, Controls N=19); the
0
central N1 (80-105 ms) is
highlighted. (B) The significant
differences across all electrodes
-2
in the time series, central N1 is
highlighted. (C) The result of a
-4
randomization test: a
ms 100 200 300 400 500 representation of p-values <0.05
B. 1
where significance had to be
maintained for a minimum of 10
ms. (D) t-values across all
electrodes with a red:blue
represents high:low.

C.
elecrtrodes

D.

204

23
 An independent sample t-test of the GFP maximum of the N1
revealed a significant difference of N1 GFP peak amplitude in
22q11.2 patients compared to controls [22q11.2 DS: 2.5040.697;
controls: 1.8830.735; mean S.D., t(df=32)=-2.5, p=0.018]. A
significant difference between groups was also observed for the
central N1 latency in response to the first click [22q11.2 DS:
85.4676.791; controls: 95.6349.737; mean S.D., t(df=32)
=3.433, p=0.002]. Analyses of Cz and GFP showed no group
difference of auditory gating at the level of the P50 (Cz: [22q11.2
DS:1.1470.48; controls: 1.2520.27; mean S.D., t(df=19)=2.09,
p=0.69], GFP: [22q11.2 DS:-0.60.127; controls: 0.1780.1; mean
S.D., t(df=22)=2.07, p=0.1]). This is illustrated by Figure.6 below.
Controls Controls
P50 Cent. N1 Lat. N1 P50 Cent. N1 Lat. N1

GFP
ms 100 400 600
1.5 200 300 500

-1.5
22q11.2 DS 22q11.2 DS

Figure.6 The P50, central N1, lateral N1 for the first and second click, separated by an
inter-click interval of 500 ms. In response to the first click, GFP amplitude of the
central N1 of patients (black) had a higher amplitude and earlier latency of the central
N1 than that of controls (red) at a significance level of p<0.05.

3.2 Pre-stimulus microstate analysis results

In both groups, the four microstate topographies showed similarity to

the microstate classes in the literature and were assigned the labels:
A, B, C, D (Britz et al., 2010; Lehmann et al., 2005; Lehmann et al.,
1998). The topographies of the classes and the distributions of the
temporal parameters are exemplified by Figure.7.

A two-way repeated measures ANOVA was conducted (results

summarized in Table.1) and revealed a significant interaction for the

24
 global explained variance of class B and class D in 22q11.2 DS
patients compared to controls. Main effects of group for time
coverage and frequency of occurrence, and main effects of microstate
class for all four temporal parameters were also observed. Post-hoc
tests showed significant group differences in the global explained
variance and time coverage of classes C and D, mean duration of
Classes B and D, and frequency of occurrence of Class D. These
results are summarized in Table.1.

A. Controls

Class A Class B Class C Class D

22q11.2 DS

B.
Global Explained Variance Frequency of Occurrence
Frequency of Occurance
Controls
0.6 3.75
0.03
* 22q11.2 DS
* Significant difference
0.4 2.5
0.02 p<0.05

* *
0.2 1.25
0.01

0.0 0
0.00
Class A Class B Class C Class D Class A Class B Class C Class D

Time Coverage (%) Mean Duration (ms)

0.6
*
120
15
0.4 * * *

80
10
0.2

0.0 405
Class A Class B Class C Class D Class A Class B Class C Class D

Figure.7 Microstate analysis results: (A) displays the spatial configuration of the four
classes of microstates x group; (B) significant ANOVA group x microstate class
interaction driven by increased global explained variance of microstate class B and
decreased global explained variance of microstate class D in the 22q11.2 DS group. A
decreased presence of microstate class C was also observed across parameters.

25
Microstate Classes A B C D
Global explained variance

22q11.2 DS mean 0.068 0.186 0.0204 0.053

S.D. 0.031 0.04 0.052 0.044
Controls mean 0.071 0.129 0.242 0.096
S.D. 0.041 0.044 0.098 0.07
ANOVA(group) F(1,32) = 2.197 p = 0.148
ANOVA(class) F(3,96) = 42.647 p =p0.000
= 0.000
ANOVA(group x class) F(3,96)= 4.233 p = .007
post hoc test, p 0.8743 0.005 0.06 0.033

Mean duration (ms)

22q11.2 DS mean 71.76 85.76 89.6 67.92
S.D. 0.658 0.81 1.6 1.124
Controls mean 71.2 79.76 93.28 74.8
S.D. 0.965 0.739 1.793 1.6
ANOVA(group) F(1,32) = 0.4 p = 0.53
ANOVA(class) F(3,96) = 27.26 p = 0.000
ANOVA(group x class) F (3,96)= 2.57 p =0.059
post hoc test, p 0.889 0.048 0.296 0.047

Time Coverage (%)

22q11.2 DS mean 0.125 0.24 0.264 0.114
S.D. 0.03 0.052 0.07 0.08
Controls mean 0.147 0.218 0.316 0.175
S.D. 0.065 0.053 0.101 0.105
ANOVA(group) F(1,32) = 9.92 p = 0.004
ANOVA(class) F(3,96) = 27.68 p = 0.000
ANOVA(group x class) F(3,96) = 1.82 p = 0.149

post hoc test, p 0.4 0.392 0.046 0.019

Frequency of occurrence
22q11.2 DS mean 1.5 2.25 2.375 1.25
S.D. 0.003 0.003 0.003 0.006
Controls mean 1.75 2.375 2.625 1.875
S.D. 0.004 0.003 0.003 0.007
ANOVA(group) F(1,32) = 6.862 p = 0.013
ANOVA(class) F(3,96) = 26.518 p = 0.000

ANOVA(group x class) F(3,96) = 0.97 p = 0.412

post hoc test, p 0.191 0.793 0.09 0.012

Table.1 Summarized results of the two-way repeated measures ANOVA conducted

on the microstate parameters of 15 patients with 22q11.2 DS and 19 controls.
Significant differences of p<0.05 are highlighted in bold.

26
3.3 Microstate and evoked activity associations

The study aimed to investigate a relationship between central N1

amplitude in response to the first click and microstate temporal
parameters but found no significant correlations between these
measures. However, Spearman rank correlation results between
microstate temporal parameters and central N1 latency showed a
positive association between global explained variance and time
coverage of microstate class A in 22q11.2 DS patients. Correlation
results are summarized in the Table.2 below.

22q11.2 DS Controls
Class A GEV mean S.D 0.068 0.031 0.0690.036
Class A Time cov. mean S.D 0.1250.03 0.1370.052
N1 latency mean S.D 85.4676.791 90.8859.851

2
Class A GEV N1 latency correlation r=0.561, R =0.315, p=0.029 r=0.115, R2 =0.013, p=0.511
2 2
Class A Time cov. N1 latency correlation r=0.592, R =0.35, p=0.02 r=0.138, R =0.019, p=0.43

Table.2 Significant positive correlations were observed for 22q11.2 DS Class A global
explained variance and N1 latency (in response to the first click), and Class A time
coverage and N1 latency, while no such correlations were present in controls.
Correlations with significance level of p<0.05 are depicted in bold.

3.4 Correlations with clinical symptoms

Both N1 latency in response to the first click and Class A time

coveragewhich correlated with one anothernegatively correlated
with thought disturbance, anxiety, bizarre behavior, unusual thought.
The global explained variance, mean durations, and time coverage of
class C positively correlated with anergia, mannerisms, motor
retardation and uncooperativeness. Results of correlations are
summarized in Table.3 in the Appendix of this manuscript.

27
IV. Discussion
In the present study, we hypothesized an increase in the amplitude of
the central N1 in response to the first click, an increased presence of
microstate class C and a decreased presence of class D in 22q11.2 DS,
and an association between these parameters. We indeed observed an
increase in central N1 amplitude and a decreased presence of class D
as expected. However, we also observed an increase of presence of
class B and a decrease of class Cthe latter being contrary to our
hypothesis. Furthermore, in the AERP to the first click, we found a
difference in central N1 latency between patients and controls and an
association between central N1 latency and the presence of class A in
patients, which correlated with clinical symptoms. Finally, we also
observed a correlation of clinical symptoms and class C. This section
is an exploration of these results.

4.1 Central N1 in 22q11.2 DS

The increased amplitude of the central N1 AERP component appears
to be characteristic of 22q11.2 DS and could be related to cognitive
and clinical phenotypes. Both the shortened latency and higher
amplitude of the N1 in 22q11.2 DS could suggest an aberrant evoked
response that might be associated with a lack of inhibitory response.
Impaired inhibition in 22q11.2 DS compared to healthy controls has
been reported in a study employing a cognitive task that measured
suppression of a startle response following an faint warning tone
before an aversive tone (Ornitz et al., 1986). This suggests a
hypersensitivityspecifically to soundin 22q11.2 DS, similar to
the implications of the present study. Another study using a cognitive
task tested the ability of 22q11.2 DS patients to inhibit the response
to a non-target stimulus, showed more parietal activation in the
patients compared to controls (Schulz et al., 2004), providing more
evidence for over-activation in the context of required inhibition.
Interestingly, our results show that both P50 and N1 auditory gating
which are associated with inhibitory filters (Freedman et al., 1987;
Jerger et al., 1992; Lijffijt et al., 2009; White & Yee, 1997)are
preserved in 22q11.2 DS. The discrepancy between an amplified N1
and preserved auditory gating could be due to a difference in the

28
neural generators responsible for the suppression of a startle
responseexpressed by a lower amplitude of the N1 AERP in healthy
subjectsand the neural correlates of P50 and N1 auditory gating.

It is also possible that the unique N1 in the AERP of 22q11.2 DS is

related to the cardiac defects that are prevalent in over 70% of patients
(McDonald-McGinn et al., 1993). It has been demonstrated that the
N1 amplitude is 10-20% larger when it is sound is synchronized with
the diastolic phase of the heart pulsewhen the heart refills with
blood, following a contraction (Sandman et al., 1984). A possibility
is that given the cardiac defects characteristic of the patient group,
there could be a link between the amplified N1 and a cardiovascular
phenomenon in 22q11.2 DS. The current longitudinal EEG study of
22q11.2 DS focuses on schizophrenia. Nonetheless, we must bear in
mind that 22q11.2 DS has a fortitude of differences from
schizophrenia and is a multi-system disorder (McDonald-McGinn et
al., 1993).

4.2 Microstate B, C and D dynamics in 22q11.2 DS

4.2.1 Class B increase in 22q11.2 DS

The increase in global explained variance and duration of microstate

class B in 22q11.2 DS patients compared to healthy controls could
imply aberrant sensory stimulation in patients. Microstate class B has
been shown to correlate with BOLD activation of bilateral occipital
areas in bilateral inferior occipital gyri, bilateral cuneus and left
lingual and middle occipital gyrus (Britz et al., 2010). These areas are
critical for visual processing, thus Class B has been associated with
the visual resting state network (Britz et al., 2010). In the present
study, participants watched a silent cartoon; as this was a constant
stimulus, it is possible that the increase in microstate class B in
patients is due to oversensitivity to visual stimulation. A visual
processing deficit in 22q11.2 DS could also explain our observation.
Visual impairments are a common clinical phenotype of the disorder
(Bassett et al., 2011) while lateral thinning of the occipital pole has
also been reported in structural studies of 22q11.2 DS (Karayiorgou
et al., 2010). Our results could be attributed to visual dysfunction, but

29
it is also possible that the increase in microstate class B in patients is
related to an interplay of sensory processing.

Though microstate class B has been associated with visual processing

(Britz et al., 2010), it could also represent other modalities of sensory
processing (Milz et al., 2016). In a recent study, Milz and colleagues
(2016) investigated changes in microstate dynamics, based on their
hypothesized functionality, and showed that class B increased during
verbalization rather than visualization. Conversely, parameters of
class A were shown to increase during visualization and not
verbalization (Milz et al., 2016). While microstate class A has been
associated with phonological processing and class B with visual
processing (Britz et al., 2010), the findings of Milz et al. (2016)
suggest that both microstate classes are involved in multi-modal
sensory processing. In relation to the results of the present study, an
increase in microstate class B in patients could be explained by a
tendency of 22q11.2 DS patients to imagine sound while watching the
silent cartoon; whether this could relate to psychosis requires further
investigation of microstate B activity in illness.

Regarding schizophrenia, reports of deviations of microstate class B

dynamics from healthy controls have been inconsistent (Rieger et al.,
2016). Two studies observed a shorter duration (Lehmann et al., 2005;
Nishida et al., 2013), while another study observed increased time
coverage of class B in patients (Andreou et al., 2014). Furthermore, a
review of microstate research in schizophrenia conducted a meta-
analysis of seven studies and showed that these effects did not
withstand correction for multiple testing (Rieger et al., 2016).
Subsequently, a clear connection between 22q11.2 DS and
schizophrenia based on the findings on microstate class B in the
present study cannot be inferred.

4.2.2 Class D decrease in 22q11.2 DS and schizophrenia

The results show that a decrease in microstate class D in 22q11.2 DS

compared to controls is robust. This decrease, in comparison to
healthy controls, appears to be stable in the group as has also been
observed at rest by Tomescu et al. (2014). In addition, a class D

30
decrease was also seen in schizophrenia in the resting state study
comparing 22q11.2 DS patients and schizophrenia patients (Tomescu
et al., 2015); this observation was line with other research showing a
decreased duration of class D in schizophrenia (Andreou et al., 2014;
Koenig et al., 1999; Lehmann et al., 2005) which suggests a link
between the populations in terms of microstate dynamics.

Functionally, class D has been associated with the central executive

resting state network involving the dorsolateral prefrontal cortex and
posterior parietal cortex (Britz et al., 2010). The central executive
network is critical for higher order cognitive processing such as
selective attention and working memory (Milz et al., 2016; Seitzman
et al., 2017), which have been observed to be impaired in both
22q11.2 DS and schizophrenia (Karayiorgou et al., 2010;
Lewandowski et al., 2007). Furthermore, studies have shown a
decreased class D duration when schizophrenia patients were self-
reporting hallucinations (Kindler et al., 2011) and a correlation
between decreased duration of class D and positive paranoid
symptoms (Koenig et al., 1999). Together, these results support a
descriptive relationship of microstate class D to compromised
cognitive processing due to central executive network dysfunction
with a possible association to psychosis in 22q11.2 DS and
schizophrenia.

4.2.3 Class C dynamics and symptoms in 22q11.2 DS

In the 22q11.2 DS population prevalence of psychotic disorder

increases in adolescence and emerging adulthood (Schneider et al.,
2014). Given that the present study focused on adolescents (12-19
years), and that most patients in the study expressed a degree of
psychosis (meaning that they had reported experiencing hallucination
or delusion) it is possible that dysfunction of saliency processing
relates to psychotic symptoms. A previous study on functionality of
resting state networks showed the salience network to be involved in
detection and orientation towards internal and external stimuli
(Menon, 2011). Likewise, increased activation in nodes of the
salience network has been shown to be associated with auditory
hallucinations (Jardri et al., 2011; Palaniyappan & Liddle, 2012).The

31
results of the present study show a correlation of class C with clinical
symptoms in 22q11.2 DS. This is in line with Tomescu and
colleagues (2014) report of a correlation between the abnormal
presence of class C at rest and scores of positive symptoms on the
Structured Interview for Prodromal Syndromes (SIPS) scale in
22q11.2 DS (Tomescu et al., 2015). Thus, these results support the
notion that increased presence of class C could be descriptive of
psychosis in 22q11.2 DS.

However, the finding of a general decrease of class C in patients

compared to controls in the eyes open task is inconsistent with resting
state observations of the group (Tomescu et al., 2014; Tomescu et al.,
2015) perhaps because due to the mechanics of the salience network
under different conditions in 22q11.2 DS. Though both resting and
the eyes open conditions are not effortful, they differ in context and
sensory stimulation, so in this sense it is not surprising that we
observe different results between the two situations. In healthy
subjects, having eyes open vs. eyes closed has indeed been shown to
affect microstate dynamics (Seitzman et al., 2017) which cautions us
from treating results in the two conditions as analogous within a
subject group. Specifically, healthy controls were shown to have an
increased occurrence of microstate class C and a decreased time
coverage of class D in the eyes open vs. eyes closed condition
(Seitzman et al., 2017), which leads us to speculate how this could be
contrasted with eyes open vs. eyes closed microstate activity in
22q11.2 DS. In relation to the stability of a decreased class D in the
patient group, we speculate that the neural activity driving this
phenomenon could in fact be stable across eyes open and eyes closed
conditions, while dynamics of class C are more vulnerable to change.

Additionally, there are several interpretations of microstate class C

which complicate the implications of our results. Britz et al. (2010)
showed that class C is correlated with what is commonly known as
the cognitive control network, saliency network, or cingulo-opercular
network, and is thought to be essential for behavioral task
performance. However, other studies have suggested it to be the
default-mode network (Milz et al., 2016; Seitzman et al., 2017).
Seitzman and colleagues (2017) showed that microstate C decreased
during cognitive tasks compared to wakeful rest, suggesting that this

32
network is task-negative which is consistent with the proposed
functionality of the default mode network (DMN). Still, Pascual-
Marqui and colleagues (2014) demonstrated that the posterior
cingulate cortex, a hub of the DMN, is involved with other microstate
generators including those of class C (Pascual-Marqui et al.,
2014). Whether class C describes the saliency network, the DMN or
a combination of both, it is possible that 22q11.2 DS patients have an
inverse dysfunction of the network i.e. it could be more activated at
rest in patients than in controls (Tomescu et al., 2014) and less in
patients than in controls during the eyes open task, but further
investigation is required to make this claim.

4.3 Microstate class A and N1 latency in 22q11.2 DS

While we expected to find a relationship between microstate classes

C and/or D with central N1 GFP amplitude we found no such
association, but instead observed a correlation between microstate
class A and N1 latency in 22q11.2 DS. The class A microstate has
been correlated with fMRI activation of bilateral superior and middle
temporal gyri, thought to be functionally important for phonological
processing (Britz et al., 2010). In the results of the present study,
auditory activation implied by the presence of class A in 22q11.2 DS
seems to be related to a longer N1 latencya latency closer to that of
healthy controlsand has an inverse relationship with clinical
symptoms. This could suggest that increased phonological processing
could play a role in improved higher order auditory processing
associated with the N1AERP response (Lijffijt et al., 2009; Naatanen
& Picton, 1987). Conversely, an early N1 response and less time
coverage of microstate A in 22q11.2 DS could be associated with
psychiatric disorder, as these both correlate with thought disturbance,
anxiety, bizarre behavior, unusual thought.

4.4 Limitations and future directions

Microstate analysis allows for us to investigate the neural networks

underlying electrical activity measured at the level of the scalp and
the association of these networks with an event related potential

33
elicited by neuronal assemblies is likely. However, the ERP has been
studied to a greater extent than microstate dynamics, thus more
research using the latter analysis must be conducted in order for us to
confidently interpret such associations. Additionally, recent studies
have raised questions about the associations of microstate classes B
and C being limited to the visual and saliency networks, respectively
(Milz et al., 2016; Seitzman et al., 2017) which complicates
interpreting the results of the present study. A technical limitation is
that the results suffered from a small sample, and it is possible that
with a larger sample results would alter in both the microstate and
AERP domains. Finally, we cannot truly testify to how microstate
activity differs in 22q11.2 DS between the eyes open and resting state
conditions without directly comparing the microstate temporal
parameters within the groups between the two conditions, thus this is
a future direction of this study.

4.5 Conclusion

Microstate dynamics and the evoked response are valuable EEG

signatures that are inherently associated and differ in health and
illness; the present study has denoted this in 22q11.2 DS. The
hyperactivity of the central N1 of the AERP in 22q11.2 DS suggests
impaired sensory processing. The association between an increased
presence of class A and central N1 latency in 22q11.2 DS may be due
to such abnormal processing. The increased presence of microstate
class B during an eyes open condition could also be descriptive of
sensory processing dysfunction in the group. A decrease in microstate
class D may be due to cognitive deficits, similar to those observed in
schizophrenia. A decrease of class C in an eyes open condition
contrasts with findings of an increased class C at rest, yet this could
be a window into the dysfunction of saliency processing in the group
across situations. The present study has reported new findings that
give direction to further investigation which may solidify these
postulations and establish reliable vulnerability markers for
schizophrenia in 22q11.2 DS.

34
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VI. Appendix

5.1 Correlations with clinical symptoms

IQ Anergia Thought disturbance Positive symptoms Anxiety Bizarre Behaviour Disorientation Excitement Hallucinations Mannerisms Motor Hyperactivity Motor Retardation Uncooperativeness Unsual Thought

Class A GEV -.2916 -.3443 -.4567 -.5108 -.3370 -.4828 -.5267 -.4181 -.4006 -.2342 -.1411 -.3218 -.2151 -.4548
p=.292 p=.209 p=.087 p=.052 p=.219 p=.068 p=.044 p=.121 p=.139 p=.401 p=.616 p=.242 p=.441 p=.088
Class A Time Coverage -.4180 -.2705 -.5201 -.4924 -.5554 -.5973 -.5774 -.4880 -.4125 -.1799 -.1525 -.1798 -.1589 -.6139
p=.121 p=.329 p=.047 p=.062 p=.032 p=.019 p=.024 p=.065 p=.127 p=.521 p=.587 p=.521 p=.572 p=.015
Class C GEV -.1021 .6486 .1728 .1701 .3800 .1710 .2283 -.2003 -.2817 .6759 -.1817 .6593 .6901 .1879
p=.717 p=.009 p=.538 p=.544 p=.162 p=.542 p=.413 p=.474 p=.309 p=.006 p=.517 p=.008 p=.004 p=.502
Class C Mean Duration .0126 .5695 .2083 .2222 .2884 .1438 .2495 -.2733 -.2023 .6112 -.1994 .5481 .6298 .1977
p=.965 p=.027 p=.456 p=.426 p=.297 p=.609 p=.370 p=.324 p=.470 p=.015 p=.476 p=.034 p=.012 p=.480
Class C Time Coverage -.2475 .5564 .0319 .0876 .1184 .0459 .2136 -.3004 -.3205 .6433 -.3057 .5814 .6738 .0465
p=.374 p=.031 p=.910 p=.756 p=.674 p=.871 p=.445 p=.277 p=.244 p=.010 p=.268 p=.023 p=.006 p=.869
N1 latency -.6516 -.2202 -.5931 -.5680 -.5330 -.5800 -.4167 -.5659 -.6946 -.0429 -.5254 -.1302 -.0179 -.5983
p=.008 p=.430 p=.020 p=.027 p=.041 p=.023 p=.122 p=.028 p=.004 p=.879 p=.044 p=.644 p=.949 p=.018

Table.3 Spearman correlation results: r, p values of correlations between temporal parameters of microstate classes A,
C, central N1 latency and psychiatric symptoms in patients. Anergia, thought disturbance and positive symptoms were
measured using the PANSS scale. Anxiety, bizarre behavior, disorientation, excitement, hallucinations, mannerisms,
motor hyperactivity, motor retardation, uncooperativeness, unusual thought were measured using the BPRS scale. IQ
was measured using on a full Wechsler Intelligence scale for children III-R (WISC-III-R) or the Wechsler Adult
Intelligence Scale III (WIAIS-III) for those 17 years of age or older. Significant correlations (p<0.05) are depicted in
red.

42