FX ENTRAIN: Perturbation of Neurodynamics Underlying Sensory Hyperarousal and Statistical Learning in Youth With FXS
Phase: NA
Conditions: Fragile X Syndrome, Autism Spectrum Disorder, Autistic Disorder, Asperger Syndrome
Sponsor: Children's Hospital Medical Center, Cincinnati

PROTOCOL TITLE: R01 FX ENTRAIN: Perturbation of Neurodynamics Underlying Sensory Hyperarousal and 
Statistical Learning in Youth with FXS 
 
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R01 FX ENTRAIN: Perturbation of Neurodynamics Underlying Sensory 
Hyperarousal and Statistical Learning in Youth with FXS 
 
PRINCIPAL INVESTIGATOR: 
Ernest V. Pedapati, MD, MS, FAAP 
Associate Professor, Division of Psychiatry and Neurology 
Cincinnati Children’s Hospital Medical Center, Division of Psychiatry  
3333 Burnet Avenue 
Cincinnati, OH 45229-3039 
Office 513.636.6553 
Fax 513.517.0860 
 
ernest.pedapati@cchmc.org 
CO-INVESTIGATORS: 
Craig Erickson, MD 
 
Professor, Psychiatry, CCHMC/University of Cincinnati (UC) 
Lauren Ethridge, PhD  
Asst. Professor, Pediatrics/Psychology, OUHSC 
Makoto Miyakoshi, PhD 
Project Scientist, CCHMC 
Stephanie Jones, PhD  
Professor, Brown University 
Laura Batterink, PhD 
 
Asst. Professor, Western University 
Steve Wu, MD  
 
Associate Professor, Neurology, CCHMC/UC 
Elizabeth Smith, PhD  
Asst. Professor, Psychologist, CCHMC 
Meredith Will, PhD 
 
Asst. Professor, Psychologist, CCHMC 
VERSION NUMBER/DATE:  
Version 1.0 // 4.03.2023 
 
REVISION HISTORY 
 
Revision # Version Date Summary of Changes 
Consent 
Change? 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Table of Contents 
1.0 
Study Summary .........................................................................................3 
2.0 
Objectives .................................................................................................4 
3.0 
Background ...............................................................................................4 
4.0 
Study Endpoints ........................................................................................8 
5.0 
Study Design and Procedures ...................................................................9 
6.0 
Sharing of Results with Subjects ............................................................. 16 
7.0 
Study Timelines ....................................................................................... 17 
8.0 
Inclusion and Exclusion Criteria .............................................................. 17 
9.0 
Local Number of Subjects ....................................................................... 18 
10.0 
Recruitment ............................................................................................. 18 
11.0 
Withdrawal of Subjects ............................................................................ 19 
12.0 
Risks to Subjects ..................................................................................... 19 
13.0 
Potential Benefits to Subjects .................................................................. 20 
14.0 
Data Management and Confidentiality .................................................... 20 
15.0 
Provisions to Monitor the Data to Ensure the Safety of Subjects ............ 22 
16.0 
Provisions to Protect the Privacy Interests of Subjects ........................... 22 
17.0 
Economic Burden to Subjects ................................................................. 22 
18.0 
Consent Process ..................................................................................... 22 
19.0 
Process to Document Consent in Writing ................................................ 23 
20.0 
Setting ..................................................................................................... 23 
21.0 
Resources Available ............................................................................... 23 
 
 
 

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1.0 
Study Summary 
 
 
 
Study Title 
R01 FX ENTRAIN: Perturbation of Neurodynamics Underlying 
Sensory Hyperarousal and Statistical Learning in Youth with FXS 
Study Design 
The proposed project includes a case-control descriptive study 
(Aims 1 & 2) and an acute, crossover clinical trial (Aim 3).  
Primary Objectives 
Aim 1: Translational neurodynamics in resting and sensation. 
Aim 2: Cognitive studies (Statistical Learning). 
Aim 3: Sensory and cognitive perturbation studies. 
Research Intervention  Closed-loop alpha auditory entrainment 
Study Population 
The study population includes subjects 5 to 15 years of age with 
Fragile X Syndrome (FXS) with full FMR1 mutations, as well as 
two age-and-sex matched cohorts of control subjects consisting 
of typically developing controls (TDC) and subjects with autism 
spectrum disorder (ASD).  
Sample Size 
180 human subjects will be recruited in total: 120 subjects total 
for case-control portion (60 FXS, 60 ASD, 60 TDC) and 60 
subjects total for clinical trial (20 FXS, 20 ASD, 20 TDC). 
Study Specific 
Abbreviations/ 
Definitions  
FXS, Fragile X Syndrome 
KO, knockout 
TDC, typically developing control 
AAE, alpha auditory entrainment 
EEG, electroencephalography 
ITC, inter-trial coherence 
ASD, autism spectrum disorder 
FMRP, Fragile X mental retardation protein 
CFC, cross-frequency power-power coupling 
ERP, event-related potentials 
TCD, thalamocortical dysrhythmia 
SL, statistical learning 
PAF, peak alpha frequency 
PPF, peak power frequency 
WT, wild type 
BCI, brain-computer interface 
WLI, word learning index 
BLE, behavior learning effect 
AM, amplitude modulated 
LSL, lab streaming layer (software component) 
GUID, global unique identifier 
NDAR, National Database for Autism Research 
NIMH, National Institute of Mental Health 
ERSP, event-related spectral power 
CNS, central nervous system 

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2.0 Objectives 
Our central hypothesis is that in Fragile X Syndrome (FXS), reduced alpha (~10 Hz) pacing and 
increased gamma (>30 Hz) noise impairs, via altered timing, the detection of sensory patterns and the 
ability to mount precise neural responses, thereby impeding cognitive functioning and contributing to 
increased sensory hypersensitivity. Our approach involves three scientific aims, which, if addressed, 
would ascertain underlying mechanisms that may alleviate sensory and cognitive impairments. 
Specific Aim 1: Translational neurodynamics in resting and sensory studies. We hypothesize 
that fine-temporal alpha and gamma neurodynamics will uncover the basis of participant- and 
subgroup-level variation that drives FXS group effects. 1A: We will acquire resting-state 
electroencephalography (EEG) and sensory auditory chirp paradigms in 40 youth (5 to 15 years old) 
with FXS and age- and sex-matched typically developing controls (TDC) and autism spectrum disorder 
controls (ASD). Neurodynamics (peak power frequency, spectral events) and biophysical modeling of 
each paradigm will be compared between groups. Neurodynamics will be assessed individually and in 
combination as predictors of psychological measures, medication effects, co-occurring 
neuropsychiatric diagnoses, and computerized cognitive testing. 1B: We aim to identify neurodynamic 
features that distinguish Fmr1-/- KO from wild type (using preexisting resting-state EEG and sensory 
auditory chirp murine data). 1C (Exploratory): We aim to identify neurodynamic features that are 
conserved between Fmr1-/- KOs and participant human subgroups based on genotype. We 
hypothesize that full mutation, non-mosaic males with FXS will share similar neurodynamics as 
the Fmr1-/- KO (knockout) mouse. Shared neurodynamics between Fmr1-/- KO and a human subgroup 
will have a high translational impact. 
Specific Aim 2: Cognitive studies. We hypothesize statistical learning (SL) will be impaired in FXS 
and the degree of SL impairment will be related to cognitive and language abilities in FXS. 2A: 
Passive SL Task will compare structured (word) entrainment via EEG intertrial coherence (ITC) 
between FXS, TDC, and ASD. 2B: Psychological measures (i.e., cognitive testing, expressive 
language sampling, and language ability) will be examined to identify key subgroups and predictors of 
neural response. As the Passive SL Task can capture an objective proxy of a cognitive process across 
a wide variety of functional levels, it will have a high impact on understanding heterogeneity in FXS. 
Specific Aim 3: Sensory and cognitive perturbation studies. We hypothesize that “bottom-up” 
alpha auditory entrainment of the corticothalamic drive will normalize the ability to mount precise 
neural responses to stimuli (increased ITC) and diminish asynchronous gamma “noise.” 3A: Acute 
perturbation with closed-loop (to individualize stimulus) alpha auditory entrainment (AAE) will be 
conducted in FXS and sex- and age-matched TDC and ASD controls (n=20 per group). AAE will be 
performed in two visits as a double-blind, sham-controlled crossover study such that each participant 
will receive AAE and sham in random order. We will assess if closed loop AAE leads to normalizing 
phase synchronization of the sensory auditory chirp and the Passive SL Task. 3B (Exploratory): We 
will determine if AAE can enhance word learning as measured by reaction time in the Active SL Task.  
3.0 Background 
Fragile X Syndrome (FXS), a monogenetic neurodevelopmental disorder, manifests varying levels of 
intellectual disability, autistic features, and sensory hypersensitivity1. To date, despite over 60 
publications of phenotypic rescue in animal models, pharmacological and behavioral treatments have 
yielded little clinical success in improving cognitive symptoms in FXS in human trials2, 3. Central to this 
discrepancy is a poor understanding of the constituent neurodynamics of averaged group effects and 
individual variability in human brain activity as related to higher-level cognitive symptomatology and 
clinical phenotype4, 5. This could, in part, account for the major discordance between mouse and 
human drug treatment effects. Additional factors including development, environment, and 
medications can further meaningfully contribute to heterogeneity. In large, published cohorts, we and 
others have identified reproducible, group-level abnormalities in resting-state EEG and sensory 
auditory chirp responses linked with intellectual disability, neuropsychiatric symptoms, and sensory 

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hyperarousal6-14. This data demonstrates that individuals with FXS do not mount precise neural 
responses to the sensory auditory chirp and, instead, show increases in “noise” in the form of 
asynchronous gamma activity10. Furthermore, a marked reduction in alpha power suggests altered 
thalamocortical function, reducing the ability to detect signal from noise in the environment and 
representing potential tractable targets for “bottom-up” entrainment. However, to translate these 
findings to effective treatments for individuals with FXS, two significant knowledge gaps must be 
addressed: 1) how does individual variability in neural activity in FXS contribute to core, impairing 
sensory and cognitive symptoms; and 2) could individually targeted, external entrainment of this neural 
activity improve sensory and cognitive function?  
Significance of Aims 
Constituent Neurodynamics of Alpha and Gamma Alterations (Aim 1):  
Despite being a single-gene disorder, FXS patients exhibit market variability in EEG, molecular, 
and behavioral phenotypes compared to the Fmr1-/-KO mouse model6, 15-18. This high variability 
is observed within and across males with full mutation (mosaic and non-mosaic) and females 
who, due to an X-linked disorder, exhibit obligate mosaicism7, 15, 19, 20. Though a link between 
Fmr1-/-KO and males with full mutation FXS can be predicted based on suspected Fmr1 
expression21, the degree to which they share conserved physiology is yet unknown. Identifying 
parallel neurophysiological markers between mice and a particular human subset would 
represent a significant breakthrough for precision medicine in FXS. EEG is a highly feasible 
method to postulate whole-brain hypotheses in FXS, for which there is consensus on group-
level effects in humans8-10, 14 and Fmr1-/-KO mice6, 22, 23. Therefore, Aim 1 studies will 
investigate the neurodynamic basis and biophysical modeling of alpha and gamma alterations 
in resting-state and sensory auditory chirp EEG17 to parse heterogeneity. 
Statistical Learning as a High-value Target in FXS (Aim 2): Though resting-state and sensory studies 
can provide insight into FXS physiology, there is an urgent need to develop cognitive paradigms that 
can be studied across the FXS phenotype and be broadly applicable to cognitive development. 
Children with FXS, for example, do not keep pace with the typical rate of cognitive development, and 
their skills plateau prematurely24. Statistical learning (SL) is the process by which humans extract 
patterns in sensory input, through passive exposure and without effort or conscious intention to learn. 
SL is present from birth25 and operates continuously across the lifespan to extract and flexibly update 
statistical regularities from the environment26. While SL operates across many different domains, it is 
thought to play a significant role in language learning, most notably in speech segmentation27. Pauses 
or other acoustic cues do not reliably demarcate words in continuous speech. Becoming sensitive to 
the statistical regularities between neighboring syllables may allow learners to discover word 
boundaries in speech28, 29. Given most individuals with FXS show significant, lifelong language 
impairments, including delay of first words, deficits in SL would be expected in FXS27. Confirming this 
hypothesis and determining whether intervention is effective at improving SL would have far-reaching 
clinical implications, as improving communication abilities is an almost ubiquitous goal for families of 
individuals with FXS30. 
Since SL is performed passively and can be 
measured through EEG independently of behavior, 
it can be captured in children with FXS regardless 
of cognitive functioning ability31, 32. During SL, 
complex patterns are passively extracted through 
environmental exposure. Interestingly, the neural 
acquisition of patterned data heuristically resembles 
the responses to sensory auditory chirp when 
monitored by EEG and can similarly be quantified 
using inter-trial coherence (ITC)31. In this regard, SL 
is an ideal bridge between passive EEG sensory 

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paradigm like the sensory auditory chirp and more complex but less feasible cognitive or language 
EEG paradigms (Fig. 4). 
Normalizing EEG through Alpha Auditory Entrainment (AAE) (Aim 3): The observed reproducible 
alterations in brain activity raise a key question: would normalization of targeted EEG activity lead to 
improved sensory and SL markers within the disorder? Thus far, it remains an open question whether 
the EEG alterations observed in FXS represent a physiological mechanism that maintains suboptimal 
brain states or if they simply reflect compensatory changes to other aberrant neural processes. 
Though previous reports have identified group-level correlations of EEG and behavioral data, the 
ability of AAE to “pace” alpha rhythms (10 Hz) into typical range can be directly quantified by online 
monitoring of real-time EEG. If successful, this could lead to a new approach to addressing the 
underlying cognitive impairment in FXS and thus alter the trajectory of intellectual development in this 
disorder2.  
Summary: To this end, we have developed a cutting-edge approach to study heterogeneity in EEG 
signatures in humans, explore their relevance for sensory and cognitive disturbances, and introduce 
perturbation studies to test causal inferences. Our approach encompasses three key areas that, in our 
opinion, are of critical importance to advancing the FXS field. First, we will perform fine-temporal 
analysis (neurodynamics) of resting-state and sensory EEG to capture individual variability, model 
biophysical properties, and quantify the constituent factors of known group effects. At this juncture, we 
also ascertain which subgroup of persons with FXS shares neurodynamic profiles with theFmr1-/-KO 
mouse model. Second, we will study SL in FXS as a cognitive proxy and identify neural markers of SL 
with individual and subgroup variability. Third, we propose to move beyond correlational studies to 
examine causal effect, for the first time, that if individualized alpha entrainment will lead to 
normalization of neural responses in response to sensory auditory chirp and SL word entrainment. 
Preliminary Data Justifying Proposed Approach 
The investigative team has collected the largest cohort to date of high-resolution FXS patient resting-
state EEGs (n=70; 32F; mean age= 20.5+/-10 years old; range=6 to 46) with age/sex-matched 
controls (n=71; 30F; mean age=22.2+/-10.7 years old; range=6-48). We recently conducted the first 
source analysis of resting-state EEG in FXS and found 1) decreased peak alpha frequency, 2) 
reduced alpha 
power, 3) centralized 
distribution (known 
as anteriorization) of 
"slow" alpha 
frequencies (8 to 10 
Hz), 4) theta power, 
not alpha power, is 
inversely coupled to 
gamma power in 
FXS, and 5) 
increased 
asynchronous 
gamma power (see 
Fig. 5 for 
summary)33.  
Global decrease and “slowing” of alpha activity in FXS: A peak frequency of 10 to 13 Hz (upper alpha) 
has specific functional relevance for several major physiological systems in typically developing 
individuals34-38. As a radio receiver prefers specific electromagnetic frequencies, neurons and 
oscillatory networks also demonstrate frequency preferences39, 40. The "switch" to a dominant peak 
rhythm at 4-8 Hz in FXS (Fig. 5A) and reduced global alpha power (Fig. 5B) may be insufficient to 
drive neural ensembles with an alpha preference35, 38, 41, 42. Thus, the canonical role of alpha in 

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establishing inhibition-timing windows for optimal sensory and neurocognitive processing may be 
impaired in FXS. 
The peak frequency of 10 to 13 Hz (upper alpha) is functionally important for several physiological 
systems in typically developing individuals34-38. Like a radio receiver, neurons and oscillatory networks 
also demonstrate frequency preferences39, 40. In FXS, there is a "switch" to a dominant peak rhythm at 
4-8 Hz (theta range) and reduced global alpha power, which may be insufficient to drive neural 
ensembles with an alpha preference35, 38, 41, 42. Thus, the canonical role of alpha in establishing 
inhibition-timing windows for optimal sensory and neurocognitive processing may be impaired in FXS. 
“Noisy” Elevated Gamma in FXS (Fig 5C): Asynchronous gamma oscillations (30 to 80 Hz) hold a 
special interest in NDDs because of their relation to cortical excitability23, 43, their association with 
perception and cognition44, and measurability in animal models22. The role of gamma oscillations is 
increasingly nuanced, such that precise synchrony in gamma power is associated with functional 
networks and higher-order cognition36, 45, but also that a modest degree of asynchrony or "noise" is 
normal46-48. However, asynchronous (or background) gamma power above what is typically expected 
has been associated with disordered states44 and, at a circuit level, is associated with reduced spike 
time precision and spectral leakage of spiking activities49.  
Evidence of Thalamocortical dysfunction:  
Alterations in thalamocortical activity may unify the system-level hypothesis that underlies these EEG 
findings. Thalamocortical dysrhythmia (TCD) is an electrophysiological motif observed in several 
neuropsychiatric conditions (e.g., epilepsy, Parkinson's disease, tinnitus, depression, and neuropathic 
pain)50-52 that reflects dysregulated cortical excitability. Like in FXS, patient groups that display TCD 
exhibit reduced alpha power, increased theta power, increased gamma power, and predominance of 
theta-gamma CFC. TCD-related EEG alterations have been associated with clinical features in these 
disorders. Importantly, in our dataset, abnormalities in alpha and gamma power demonstrate 
significant clinical associations with several core features of FXS, including cognitive function, anxiety, 
social communication, and auditory attention. 
Feasibility of Statistical Learning Tasks (Aim 2): Extensive work by our team (Dr. Batterink) has reliability 
demonstrated in infant and youth populations31, 53 that neural entrainment of SL can be tracked using 
EEG (see Fig. 1C, pg. 13). To date, SL has not been investigated in FXS, but is a highly salient area of 
study in FXS given the interest in language acquisition and as a cognitive proxy for learning in youth with 
FXS19, 54. Furthermore, neural entrainment is amenable to neurodynamic analysis, as embedded 
words gradually increase as the exposure progresses and predicts improvement in reaction time to test 
SL performance. 
Evidence of neural entrainment by AAE in FXS (Aim 3): To date, neural perturbation, or entrainment to 
enhance cortical alpha activity, remains unexplored in FXS. The presence of hyperacusis (and EEG 
evidence of sensory hyperarousal) led the investigative team to consider using rhythmic alpha-frequency 
auditory signal to modulate cortical activity. We reasoned that an auditory entrainment signal could drive 
alpha oscillatory activity at both subcortical and cortical levels because encoded acoustic stimuli 
ultimately reach the temporal lobes via a thalamic intermediary (medial geniculate nucleus). Auditory 
entrainment has been used effectively as a neural probe, both for short-term suppression of tinnitus55, 56 
as well as eliciting physiological changes in EEG parameters57. Though audio entrainment has mixed 
evidence for durable effects as a therapeutic modality or cognitive enhancement58, it has been shown in 
some clinical populations to have robust immediate or short-term physiological effects that can be 
measured by EEG59-62. We hypothesize that enhancement of alpha activity via AAE may activate neural 
circuits that have an alpha preference, and this enhancement can be observed by increased number of 
transient alpha spectral events, normalization of sensory auditory chirp response (increased ITC and 
decreased asynchronous gamma), and enhancement of SL (as measured by increase in word learning 
ITC). Closed-loop implementation: We have integrated AAE into a closed-loop perturbation paradigm in 
which alpha power can "self- correct" based on real-time responses. Closed-loop perturbation is a 
powerful framework for testing circuit hypotheses of complex neural phenomena and has emerging use 

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in clinical settings for epilepsy63, Parkinson’s64, and Tourette’s disorder65. A neural closed-loop system 
would ideally account for individual variability in response by automatically achieving the desired target 
by modulating the input signal based on empirical feedback66. 
AAE Feasibility data: Our approach is ideal for children as it is delivered at hearing threshold using 
headphones and the perturbation can be rapidly modulated in real-time. Unlike other non-invasive brain 
stimulation (e.g., TMS or TDCS), our approach is specifically tailored to a "bottom-up" approach by 
driving cortical oscillations through thalamocortical drive via auditory input (which we hypothesize is 
impaired in FXS). We developed a novel AAE stimulus (“baseline AAE”) that creates a differential 
rhythm between the left and right ear starting at high theta range (7 Hz) through high alpha (13 Hz) in 2 
Hz steps on a 500 Hz sine carrier tone.  
AAE modifies alpha power in FXS: To demonstrate feasibility and obtain pilot data we performed 
baseline AAE with EEG on 8 subjects (TDC: 6M, 7M, 8F, 20M; FXS: 15F, 28M, 38M, 48M). All subjects 
completed the paradigm without any adverse events. The eventual goal of baseline AAE is to identify a 
starting frequency for closed-loop perturbation to account for baseline individual variability. Our pilot 
findings provide supportive evidence of our central hypothesis of alpha entrainment that may provide 
compensatory action within the FXS brain. 1) We demonstrate a high level of neural response variability 
to AAE (controlled by sham) in eight subjects (Fig. 10A), providing rational for the closed-loop approach 
and further emphasizing the importance of capturing individual variability in therapeutic development. 2) 
We show evidence of AAE’s effects on resting-state EEG in a 15-year-old FXS female, wherein AAE 
appeared to increase alpha power and decrease theta power over sham AAE (Fig. 10B Left). 3) In the 
same subject, using peak power frequency analysis (PPF) we identified both an exogenous leftward-
shifted “alpha” peak and increased alpha peak power between 9 to 11 Hz (Fig. 10B Right). 4) Using 
causality analysis (direct directed transfer function), we observed evidence of AAE information outflow 
(phase-based and controlled via sham) from the temporal lobes to frontal, central, and occipital regions 
in a 7-year-old male (Fig. 10C)  
4.0 Study Endpoints 
Aim 1: Sensory Auditory Studies 
Primary outcomes of interest: 1) Neurodynamic features (peak power frequency, event rate, 
event duration), 2) 40 Hz ITC of the sensory auditory chirp, 3) Asynchronous gamma power. 
Aim 2: Cognitive Studies 
2/Hz dB)
Power AAE-Sham (uV
-2
0
2
4
7 Hz-SHAM
9 Hz-SHAM
11 Hz-SHAM
5
10
15
4
-2
0
2
4
4
5
10
15
Frequency (Hz)
Frequency (Hz)
target Hz
13 Hz-SHAM
AE - Sham
5% FDR adjusted
13 Hz AAE
Sham
10
20
-24
-22
-20
-18
-16
-14
-12
-10
*
*Theta
Alpha
Power (10*log10uV2/Hz dB)
7 Hz
9 Hz
11 Hz
13 Hz
Direct Directed Transfer
Causality Function (dDTF)
AAE  > Sham
FDR < .001
Peak-power sorted time
2 
4 
8 
13
20
30
Frequency (Hz)
-26
-22
-18
-14
-10
-6
dB
1. Endogenous ‘alpha’
at 6 Hz
3. emergence of
“mu” rhythm
2. increased peak
alpha frequency power
B. Alpha Enhancement in FXS youth
A. AAE Response
C. Phase entrainment
Fig. 10: Neural Entrainment by AAE. A. As expected, AAE resulted in high level of interindividual variability between stimulus frequencies. AAE power 
following subtraction of Sham. In our design, a starting frequency would be selected based on maximum response and then AAE would proceed in a 
closed-loop fashion individualized to each participant. B. Left: 13 Hz AAE vs. sham resulted in an increase in alpha power and a decrease in theta power 
in a 15-year-old female with FXS (paired-t test).  Right: Peak Power Frequency (PPF) analysis following AAE baseline show endogenous “alpha peak” 
(~6-7 Hz) and AAE induced alpha peak (~9-11 Hz). Interestingly, the sensorimotor mu rhythm appeared following stimulation, suggesting normalization of 
alpha rhythms. C. Evidence of AAE information flow (phase-based) from L temporal lobe to frontal, central, and occipital regions using causality analysis. 

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Primary: Word-learning Index (WLI) for the SL passive exposure task, i.e., EEG phase-
synchronization at 1.1 Hz (word rate) compared to 3.3 Hz (syllable rate) over the exposure period. 
Exploratory: Learning Effect (BLE) of the SL active performance task. 
Aim 3: Neural Perturbation Studies 
Primary: 1) AAE versus sham change in 40 Hz ITC, and 2) WLI during SL passive exposure task. 
Exploratory: AAE vs. sham effect on reaction time (indicated by Behavior Learning Effect) during 
SL active performance task. 
5.0 Study Design and Procedures 
5.1 Study Design 
Aims 1 & 2, combined, consist of a case-control study that will be completed over two visits lasting 
approximately 3-4 hours each, including: medical and psychological assessments, neurocognitive 
testing, and EEG/event-related potentials (ERP) procedures. Visits one and two may be completed up 
to three months apart. Following completion of Aim 1 and 2 studies, subjects and their guardians may 
be asked to participate in Aim 3.   
Aim 3 (Intervention) consists of a two-visit, randomized controlled, crossover acute perturbation study 
to study the effect of AAE or sham stimulation on 1) the sensory auditory chirp (ITC and asynchronous 
gamma) and Passive SL Task (word learning ITC) and 2) in sufficiently cooperative participants, 
reaction time responses of an exploratory Active SL Task (word learning). AAE will be performed in 
two visits as a double-blind, sham-controlled crossover study such that each participant will receive 
AAE and sham in random order. For Aim 3, we estimate two 4-hour visits separated by at least a 1-
week washout.  
FX ENTRAIN Studies flow: 
 
5.2 Procedures 
The number of research visits for each subject may vary depending on subject age/functioning, 
tolerability of procedures, and family schedule. The research team will work to always fit the needs of 

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the subject/family. In line with this, subjects, caregivers, or parents may be given the option to complete 
appropriate surveys or questionnaires electronically through Redcap, over phone/video call with a 
member of the study team, or from home and mailed back to the research team. We may not administer 
all of the measures, assessments, or procedures, listed below. Measures administered will be at the 
discretion of the PI. If any data is unable to be collected, this will not be considered a protocol violation, 
but will be noted in the study chart. When applicable, assessments and measures previously collected 
from related, linked studies conducted by the Neurobehavioral Research Team and associated PIs can 
be transcribed and used for this study to reduce patient burden and the possibility of test-retest effects. 
Clinical data may include, but is not limited to, previous behavioral, neuropsychological, or other testing, 
and the results of any pertinent laboratory tests including previously conducted genetic chromosomal or 
microarray testing, vital signs, medication history, imaging history, CBC, metabolic panels, lipid panel, 
and other blood testing. 
Standardized Measures and Assessments 
Participants may complete the following: 
1. Brief medical history and physical exam to determine any exclusion criteria and 
medication. Medication history will contain medication details including duration taking the 
medication, time of last dose, history (with start and stop dates) of discontinued medications, and 
any non-pharmaceutical treatments such as herbals or complementary medications. Physical 
exam may include height, weight, head circumference, vitals and CNS data. 
2. Hearing and vision tests to assess adequate hearing threshold using conventional threshold 
measurements from 0.25 to 8 kHz with simple behavioral responses, and vision tests to confirm 
corrected far visual acuity of less than 20/40.  
3. Social Communication Questionnaire (SCQ)67 which is a screener for autism spectrum 
disorder (ASD). TDC controls must score ≤8 to be included in the study and ASD controls must 
score < 15 to be included in the study.  
4. Autism Diagnostic Observation Schedule-2 (ADOS)68 will be completed to assess for co-
morbid ASD in the FXS cohort, and to confirm ASD diagnosis in the ASD cohort. All ASD subjects 
and only FXS subjects scoring ≥ 12 on SCQ will complete the ADOS.  
5. Mini International Neuropsychiatric Interview Kid (MINI-KID)69: Structural diagnostic 
interview to assess call co-occurring disorders in all subjects.  The standard MINI Kid assesses 
the 30 most common and clinically relevant disorders or disorder subtypes in pediatric mental 
health.  
6. Stanford Binet IQ Scale-5th Ed (SB-5)70. to provide a Full Scale, Verbal, and Nonverbal I.Q.  
7. Vineland Adaptive Behavioral Scales- Third Edition71, to assess adaptive skills.  
8. Expressive Language Sampling (ELS), conversation, and narrative19 to assess naturalistic 
language abilities and heavily used in FXS.  
9. Clinical Evaluation of Language Fundamentals (CELF-5)72: Comprehensive evaluation of 
language abilities to determine the level or levels of language (e.g., semantics, morphology, 
syntax, and pragmatics). 
10. Parent-report measures of psychological functioning: Aberrant Behavioral Checklist (ABC)73; 
Anxiety, Depression, and Mood Scale (ADAMS)74; Sensory Profile75; Social Responsiveness 
Scale (SRS-II)76; Repetitive Behavior Scale-Revised (RBS-R)77; Pediatric Anxiety Rating Scale 
(PARS)78; Pediatric Quality of Life (PQL)79.  
11. NIH Cognitive Toolbox80 for neurocognitive and neuropsychological skills. 
 
 
 
Table 1: Detailed list of medical and standardized study assessments by cohort 
 

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Domain  
Assessment  
FXS  TDC  
ASD  
Remote 
Administration 
Online 
Survey 
Medical  
Medical 
assessment 
(history, 
concomitant 
medication 
evaluation)  
X  
X  
X  
X 
  
  
Physical 
(height, weight, 
head 
circumference, 
vitals, CNS data) 
X  
X  
X  
  
  
  
Hearing and 
vision screener  
X  
X 
X  
  
  
Diagnostic  
Social 
Communication 
Questionnaire 
(SCQ)  
X  
X  
X  
X 
X 
Autism 
Diagnostic 
Observation 
Schedule 
(ADOS)  
  X*  
  
X  
  
  
Mini 
International 
Neuropsychiatric 
Interview Kid 
(MINI-KID)  
X  
X  
X  
X 
  
Cognitive/Adaptive  
Stanford-Binet 
Intelligence 
Scale, 5th 
Edition  
X  
X  
X  
  
  
Vineland 
Adaptive 
Behavior Scale 
3rd Edition 
X  
X  
X  
X 
  
Language  
Clinical 
Evaluation of 
Language 
Fundamentals 
(CELF-5)  
X  
X  
X  
  
  
Expressive 
Language 
Sampling (ELS) 
X  
X  
X  
  
  
Psychological   
Aberrant 
Behavior 
Checklist (ABC)  
X  
X  
X  
X 
X 
Anxiety, 
Depression, and 
X  
X  
X  
X 
X 

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Mood Scale 
(ADAMS)  
Sensory Profile  
X  
X  
X  
X 
X 
Social 
Responsiveness 
Scale (SRS-II) 
X  
X 
X  
X 
X 
Repetitive 
Behavior Scale-
Revised (RBS-R)  
X  
X  
X  
X 
X 
Pediatric 
Anxiety Rating 
Scale (PARS)  
X  
X  
X  
X 
X 
Pediatric 
Quality of Life 
X  
X  
X  
X 
X 
Neuropsychological  
NIH Cognitive 
Toolbox  
X  
X 
X  
  
  
* Only administered to FXS subjects with SCQ score ≥12  
Detailed schedule of procedures by aim/visit 
 
 
Neurophysiology Tasks 
Table 2: Schedule of neurophysiology and neurocognitive procedures 
 
Aim 1 (Visit 1): 
Sensory Auditory Studies 
Aim 2 (Visit 2): 
Cognitive Studies 
Aim 3 (Visits 3 & 4):  
Closed-Loop Neural 
Perturbation Studies 
Resting State EEG  
Resting State EEG 
Resting State EEG  
Sensory Auditory Chirp  
Passive SL Task  
AAE1 Baseline   
Auditory Steady State 
Active SL Task 
Sensory Auditory Chirp 
AAE1 Baseline  
AAE1 Baseline 
Passive SL Task 
 
 
Active SL Task 
1AAE, Alpha Auditory Entrainment Paradigm 

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EEG/ERP Procedures 
EEG/ERP may be used to assess the electrophysiologic correlates of exposure to sensory stimuli (i.e. 
auditory/, visual, tactile) and/or during behavioral computerized testing. Recordings are acquired using 32 
up to 256-channel cap (HydroCel Geodesic Sensor Net) with an amplifier. Data are recorded at a sampling 
rate of 1000 Hz with saline-based electrodes. Acoustic stimuli are generated and presented using 
MATLAB (MathWorks, Natick, MA) and synchronized with a pulse to mark the onset of each stimulus 
Video may be recorded to accurately identify non-brain artifact contamination of the recording. In order to 
facilitate accurate EEG electrode locations on the scalp during analysis any subject that receives an EEG 
may be photographed to determine electrode positions during recording; this may include a 5-10 minute 
procedure in which the EEG net and electrode positions are captured using photogrammetry software and a 
stereoscopic camera. Data will be used to generate a numerical coordinates file. All EEG collection will be 
completed by trained research staff members. Our team developed systematic behavioral preparation 
including social stories, visual schedules, and demo nets/parent training to optimize data collection. In 
our experience, >95% of study eligible individuals with FXS provide usable EEG data following our study 
protocols. To maintain cooperation with study procedures, resting and sensory EEG is performed while 
watching a standardized silent cartoon. Behavioral tasks will use response button boxes. 
1. Resting state EEG: Continuous data will be recorded for 5-7 minutes. 
2. Sensory Auditory Chirp: "Chirp" stimuli will be delivered through headphones/speakers with a 
1000-Hz carrier tone amplitude modulated (AM) by a chirp sinusoid linearly increasing in 
frequency from 0–100 Hz over 2000 ms. One-to two-hundred stimuli will be separated by a 1.5-
2 sec inter-trial interval, for 7.5-12.5 minutes of testing.  
3. Sensory Auditory Steady State: As an exploratory outcome, we may test stability of 
synchronization at alpha (10 Hz) frequency and its relation alpha power phase. Stimuli will consist 
of white noise amplitude modulated by a sinusoid for 3 sec at 10 Hz. Fifty trials at each frequency 
(100 trials total) will be randomly presented and separated by a 1.5-2 sec inter-trial interval, for 
8.3 minutes of testing.   
4. Alpha Auditory Entrainment (AAE) Baseline: The AAE was generated in MATLAB 2019b with 
a 500 Hz sine carrier wave. We had five total conditions including silence (no stimulus), sham 
(500 Hz both ears), and linearly increasing alpha frequencies of 7 Hz (507 Hz in right ear), 9 Hz 
(509 Hz in right ear), 11 Hz (511 Hz in right ear), 13 Hz (513 Hz in right ear). The total stimulation 
length is twelve minutes with one minute of stimulation of each frequency in pseudorandom 
order.   
5. Dynamic AAE during closed-loop perturbation: The requested frequency differential will be 
generated by a custom function in MATLAB to deliver AAE in the requested .5 Hz step. This 
stimulus will last for 60 seconds until the next iterative feedback. Combining AAE with paradigm: 
The AAE stimulus will be played back at background noise level alongside either the sensory 
auditory chirp or SL tasks. There is considerable literature on the combination of entrainment 
beats with other auditory stimuli (speech, music) maintaining the physiological response81-83. 
6. Sham AAE: The sham stimulus was generated in MATLAB with a 500 Hz sine carrier tone. The 
sham AAE condition will mimic the closed-loop AAE condition detailed above, however only the 
carrier stimulus (500 Hz) is mixed with the paradigm audio during sensory auditory chirp or SL 
tasks.  
Eye Tracking 
Participants may be monitored or asked to make specific eye/eyelid movements. Behavioral and 
emotional tasks may be used in conjunction to monitor eye movements. This task involves the subject 
to look at a sequence of varying emotional faces and social sequences. The resulting ocular movements 
may be recorded using a Tobii Eye Tracker (Tobii Technology AB, Danderyd Sweden) or similar 
scanner-compatible technology. 

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Statistical Learning Tasks  
Assessment of SL includes a 1) Passive SL Task and 2) Active SL Task. This approach allows us to 
capture data on all participants regardless of adaptive functioning. The Passive SL Task uses an EEG-
based measure of neural entrainment involving ITC31, 53 to calculate Word Learning Index (WLI). The 
Active SL Task based measure uses reaction time to estimate a behavioral measure of SL, the 
Behavioral Learning Effect BLE32. The tasks may be altered (e.g., speed reduced, additional instruction 
or practice given) to increase feasibility for participants.   
Procedure Overview (all SL tasks) 
Participants will be seated in front of a computer monitor with headphones compatible with EEG cap. If 
a subject is not able to tolerate headphones, alternative methods like speakers may be used instead. 
To facilitate cooperation and reduce motion artifact, standardized, silent non-social videos will be 
played during the task. Stimuli (see Fig. 1): Twelve syllables are combined to make four, three-syllable 
pseudowords. This miniature synthetic language resembles natural language as (1) transitional 
probabilities are higher for neighboring syllables within words than across word boundaries and 2) a 
given syllable may be present in more than one word. For example, a transitional probability of 1.0 for 
a syllable pair such as "vinay" indicates that every "vi" in the stream was followed by "nay." SL occurs 
as the brain "discovers" hidden words in the speech stream by becoming sensitive to these linguistic 
patterns through repeated exposure. 
Passive SL Task: Each participant will be presented with both a structured and random stream of 
syllables. The structured stream will consist of the four generated words, concatenated in 
pseudorandom order without any pauses or acoustic cues between words (e.g., vinayfeetidomoo…). 
Each word will be presented 200 times, for 800 total word presentations.  Order will be constrained to 
avoid successive syllable and word repeats. The random stream contains the same total number of 
syllables, which are ordered pseudorandomly to avoid syllable repetitions but will otherwise not follow 
any hidden structure. Words will be presented at a rate of 1.1 per a second (thus, syllable rate is 3.3 
Hz), resulting in 10 minutes of each structured and random continuous stimuli. As previously published, 
the expected outcome of this study is an increase in EEG phase-locking at 1.1 Hz over the exposure 
period—present in the structured condition only—indicative of gradual word discovery and successful 
word segmentation resulting from statistical learning. 
Word-learning Index (WLI) for Passive SL: WLI is the primary EEG outcome measure for the exposure 
task and, similar to our sensory studies, is based on intertrial phase coherence (ITC). As the participant 
"learns" the four words in the structured stream (i.e., uses statistical probabilities across neighboring 
syllables to segregate the speech stream), their EEG typically shows increased phase-locking 

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(increased ITC) at the word presentation rate (1.1 Hz) and decreased phase-locking at the syllable 
presentation rate (3.3 Hz). Thus, patterns of EEG phase-locking, corresponding to a shift in processing 
from raw syllable units to cohesive words, reflect gradual statistical learning in the brain. The effect can 
be quantified by creating a ratio of the ITC for words versus syllables, as follows:   
WLI = Intertrial Coherence word rate / Intertrial Coherence syllable rate  
A higher WLI indicates a relatively stronger response to tri-syllabic nonwords compared to raw syllables, 
reflecting stronger word segmentation due to statistical learning. The WLI predicts subsequent 
performance on behavioral tests of statistical learning and increases over time in the structured condition 
only, and not in the random condition31.  
Active SL Task (Exploratory): Following the exposure task, each participant may attempt to perform a 
reaction-time based task that provides a behavioral index of SL. Participants will be instructed to press 
a button when they hear a given target syllable (e.g., "moo"). Prior to the main task, the participant may 
complete 10 practice attempts on non-experimental syllables to assess feasibility. The four words from 
the exposure task will then be presented a total of 20-45 times per word across a total of five, one-
minute blocks, each corresponding to a separate target syllable. Within each one-minute block, each of 
the six words will be repeated 4-9 times. A visual reminder of the target (e.g., a picture of a Cow for 
"moo") will be displayed for the entirety of the block. Targets presented within later positions within a 
nonsense word, (i.e., those syllables that are more predictable) elicit faster reaction times than less 
predictable, word initial syllables, providing a reaction-time based measure of statistical learning. Thus, 
the Behavioral Learning effect can be quantified as follows:  
BLE (in ms) = (Reaction time target syllable position 1)-(Reaction time target syllable position 3)  
Larger BLE values indicate greater proportional facilitation to predictable words in the stream, indicative 
of stronger SL at the individual level. Group comparisons can be facilitated by dividing BLE by the 
Reaction time target syllable position 1 to normalize to an individual's baseline reaction time. Larger BLE values 
indicate greater proportional facilitation to predictable words in the stream, indicative of stronger SL at 
the individual level. 
Interventional Agent: Alpha Auditory (Neural) Entrainment 
Randomized, 
sham-controlled 
acute 
neural 
perturbation 
studies will test the hypothesis 
that enhancing alpha oscillations 
in 
FXS 
will 
enhance 
thalamocortical drive in FXS and 
normalize 
observed 
neuro-
oscillatory alterations in sensory 
and cognitive paradigms. As this 
is a highly novel approach, we 
have attempted to reduce the 
complexity of the protocol to be 
feasible in youth and simplify 
interpretation for future chronic 
dosing studies. 
Procedure Overview 
Three segments (approximately 
15 
minutes 
each) 
will 
be 
conducted to 1) identify the baseline AAE, 2) conduct closed-loop perturbation sensory studies, and 3) 

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conduct closed-loop perturbation SL studies. Similar to Aim 1 and 2 studies, subjects will be seated 
comfortably and allowed to watch silent, non-social videos during setup, segment 1, and the passive 
portion of segment 2 to facilitate cooperation and reduce movement artifact. Following the first segment, 
the subject will be offered a 5 to 10-minute break. 
Baseline AAE (Segment 1): To avoid extensive "frequency finding" during the closed-loop segments we 
have opted to perform a linear increasing auditory entrainment paradigm to identify an optimal starting 
frequency. We will record 3 to 5 minutes of resting state EEG prior to the AAE baseline paradigm. The 
brain computer interface (BCI) will provide real-time detection of peak-power frequency and magnitude 
of the target frequency range at each frequency step and will be recorded with subject data. Following 
completion of the baseline AAE paradigm for each subject, the investigator(s) will evaluate output curves 
and determine optimal starting frequency. Segment 1 is performed during AAE or sham treatment 
conditions. 
Closed-loop segments 2 and 3: Paradigm procedures for both sensory auditory chirp and SL paradigm 
will be identical to Aims 1 and 2, with the addition of the AAE stimulus (either active or sham) to the 
paradigm audio. As the SL Tasks syllables can be varied, the subjects will learn a new “miniature” 
language on each visit. In the closed loop condition, prior to starting paradigm sixty seconds of the 
selected baseline AAE will be delivered via earphones before the start of the paradigm to capture 
sufficient data for feature feedback. Following initiation of the paradigm, every 60 seconds a moving 
average of peak power frequency will be broadcast via LSL to MATLAB for adjustment of entrainment 
frequency. If the peak power frequency is less than the target frequency entrainment stimulation will 
advance by .5 Hz. If the peak power frequency is higher than the target frequency, the entrainment 
stimulus will decrease by .5 Hz. If the peak power frequency is within the target, no change in frequency 
will be performed. As in Aim 2, all subjects will receive the SL exposure task, however, subject feasibility 
will be assessed for completion of the SL performance task. Following completion of paradigm, subjects 
will be allowed to watch a video of their choice (with sound) as the EEG cap is removed to facilitate 
cooperation and serve as a reward. 
5.3 Blindness and Procedure for Breaking the Blind 
This is a double-blind, sham-controlled study. Investigators, study staff, and study subjects will be 
blinded to the randomized study treatment assignments. Unlike drug studies, the “treatment” in this 
study is the AAE stimulus (alpha auditory entrainment) versus Sham (carrier frequency alone) without 
an alpha entrainment rhythm. Each subject will get either AAE or sham, but the order will be 
randomized and blinded. A balanced, coded randomized list of treatment orders for sixty subjects will 
be generated via software. The key list will be kept with a designed study personnel (who is not 
involved in running subject visits or data analysis). The blinded list will be coded with BCI software to 
deliver the correct treatment order of Sham or AAE. As the stimulus will be delivered via headphones 
or speakers at background noise levels and generate identical triggers study personal will remain 
blinded until after study completion. Though we do not anticipate any need for an emergency 
unblinding, it will only be considered in situations where the knowledge of the treatment code has an 
impact on the planned treatment of the emergency. Once the randomization code is broken for a 
subject, he/she must be withdrawn from the study. The investigator must record the reason for the 
emergency unblinding in the source documents and recorded in the electronic database. 
5.4 Protection Against Risks (all aims) 
All experimental procedures will be carried out in a setting and among staff who are carefully trained 
and experienced in working with individuals with developmental disabilities including FXS and ASD. All 
human research procedures take place on the clinical campus of CCHMC. In addition to the on-site 
presence of an attending physician who is PALS certified, the clinical resources of CCHMC will be 
available in the case of an adverse event which includes access to AED equipment and the 
Emergency Department. See Risks to Subjects section for detailed precautions against risks. 
6.0 Sharing of Results with Subjects 

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Findings from assessments will be made available to agents of the participants (parents, schools, 
treating psychologists, physicians) only with the expressed, written consent of the parents/guardians 
and if appropriate for the study participant. 
The study investigator team is joined by a pediatric neurologist (S.W.) with expertise in clinical EEG 
and brain stimulation and extensive collaboration with the P.I. on pediatric TMS studies84-89. Since 
ASD patients have a higher risk for epilepsy than the general population, the neurologist's role will be 
to review all research resting-state EEGs for abnormalities, including evidence of seizure activity. If 
any abnormalities are found, these findings' clinical relevance and urgency and the need for a 
neurological exam will be discussed with the principal investigator. The findings will be directly 
discussed with the family with appropriate clinical referral. In addition, any features of clinical 
significance within the EEG recordings will be considered in subgroup and co-variate analysis. 
7.0 Study Timelines 
Individual subjects’ duration of participation in this study is estimated to be approximately 16 hours 
over the course of up to two years, with up to three months of separation allowed between visits 1 and 
2 (descriptive study), and 3 and 4 (clinical trial), respectively. It is expected that participants’ duration 
of participation will be significantly shorter than is stated above, however, considering many FXS 
families travel great distances to be seen in our lab clinically, high flexibility in scheduling is imperative 
for ensuring equal opportunity to those who wish to participate in research. 
We plan to enroll all study subjects by end of year 4 and estimate the primary analyses will be 
completed by end of year 5.  
8.0 Inclusion and Exclusion Criteria 
Inclusion Criteria (all aims): 
FXS 
ASD 
TDC 
Aged 5-15 years, inclusive 
X 
X 
X 
Patient has full FMR1 mutation confirmed by genetic testing 
X 
  
  
Score ≤ 15 on SCQ screen 
  
X 
  
Have documentation of ASD diagnosis 
  
X 
  
Have no known genetic mutation 
  
X 
X 
Be in good health per investigator 
  
X 
X 
Patient has met normal developmental milestones 
  
  
X 
Patient has no family history of heritable neuropsychiatric disorders 
  
  
X 
Patient has an IQ greater than 85 on the Stanford-Binet 
  
  
X 
Score ≤8 on an SCQ screen 
  
  
X 
Exclusion Criteria (all aims): 
FXS 
ASD 
TDC 
Patient has auditory or visual impairments that cannot be corrected 
X 
X 
X 
History of substance abuse or dependence within the past 6 months 
X 
X 
X 
 
Vulnerable Populations 
• 
Inclusion of women and minorities: We will include males and females in numbers consistent 
with the distribution expected in the clinic populations. We will not exclude any participant 
based on race or ethnicity. The distribution of minorities will be consistent with the distribution 
expected in Cincinnati. 
• 
Inclusion of children: A central aim of the proposed research is to evaluate the underlying 
mechanisms responsible for brain development in children with FXS. This can only be done by 
studying affected individuals in that age group. The minimum age of 5 was chosen because of 

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the level of cooperation required to complete procedures, and the upper age limit of 15 was 
chosen because of common age-related changes in behavior and brain function beyond that 
age.  
• 
Persons with Cognitive Impairment or other limited decision-making capacity: To be inclusive 
and most generalizable in our study findings, and because some elements of our protocol can 
be completed with minimal cognitive requirements, such as event-related potential (ERP) 
monitoring of cortical responses to tones, we will recruit any study-eligible patient with Fragile X 
Syndrome regardless of level of cognitive disability into as many study components as they can 
complete. We will recruit all FXS patients who we believe can complete study procedures. 
9.0 Local Number of Subjects 
The target enrollment for the case-control portion (Aims 1 & 2) is 120 subjects and 60 subjects for the 
clinical trial (Aim 3), for a total of 180 subjects across all aims. We plan to recruit a total of 60 subjects 
with Fragile X Syndrome with full FMR1 mutations (>200 CGG repeats; at least partial FMR1 gene 
methylation). Of these FXS subjects, 40 will be recruited for the case-control portion and 20 will be 
recruited into the clinical trial. The overall project will recruit an additional two cohorts of control 
subjects for a total of 60 IQ-, sex-, and chronologically aged matched non-syndromic (i.e., idiopathic) 
ASD controls, and 60 sex-and chronologically aged matched TDCs. Following completion of the case-
control portion, subjects and their guardians may be asked to participate in the clinical trial portion of 
the study.  
Retention Plan: We do not anticipate a high drop-out rate since almost all testing is non-invasive, and 
each portion of the study (case-control and clinical trial, respectively) can be completed in 2 visits, with 
ample time allowed between visits. Thus, we expect close to 90% retention of all recruited subjects. 
Subjects unable to complete study procedures or who fail to consent/withdrawal following enrollment 
will be replaced to maintain our enrollment target of 180 total subjects. 
10.0 Recruitment 
Recruitment Methods and Materials 
Potential participants may be recruited for the study at CCHMC, from the general public, and at special 
community events or lectures. In some instances, subjects who have consented to be contacted for 
future studies may be sought if they qualify for this study. Participants may also be recruited from local 
and national databases intended for recruitment, EPIC queries, and OCTR database queries. 
Fragile X subjects will be recruited from the Cincinnati Fragile X Research and Treatment Center by 
treating physicians and their staff. The clinical team will present the project to patients and family 
members when they are at our clinics or treating physicians may contact them directly about their 
interest in participation.  
Participants with a history of Developmental Disability will be recruited from the CCHMC 
Developmental Disabilities Psychiatry Service (inpatient and outpatient services), the CCHMC Division 
of Developmental and Behavioral Pediatrics outpatient clinics, other CCHMC outpatient clinics, 
CCHMC inpatient units, at special events and from the general public.  
The study will be advertised in hospital/clinic and general public areas via print, email, electronic and 
social media. The Cincinnati Fragile X Research and Treatment Center will be sending out a monthly 
newsletter providing resources, research findings, upcoming events, and research opportunities to 
families who have expressed interest in clinic/research; information about research opportunities will 
also be distributed in the form of a brochure. All recruitment materials mentioned above will be IRB-
approved prior to send-out. 
Interested families or potential participants may contact the research team directly by the phone 
number or email address given in the advertisements, at which time a research coordinator will follow 
up by phone or email. When individuals contact our research staff or provide consent to be contacted, 

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we will provide them with basic information regarding study procedures and aims, inform them that this 
research is voluntary, and answer any questions about the study. Family and medical history will be 
reviewed in person or over the phone to determine study eligibility. If no exclusionary factors are 
identified in the review of family and medical history, then subjects will be scheduled for a meeting to 
obtain informed consent. 
Potential participants and their families may be given the option to have a practice cap/beanie mailed 
to them to aid with sensory familiarity in preparation for a potential future visit. For some participants 
with a history of Developmental Disability, the optional EEG tasks may present an uncomfortable 
sensory experience, especially to those unfamiliar with the EEG cap. To allow for adequate practice 
and preparation time, this option may be presented before the potential participant has been 
consented to the study. The practice beanies fit snugly around the head, offering a similar sensation to 
a real EEG cap. If the family already owns a similar cap the subject can practice with they may be 
asked to use that instead. The caps will not be used to screen subjects and are only being provided to 
increase subjects’ comfortability with EEG. Subjects who opt to receive a practice cap are not required 
to practice wearing the cap prior to the first visit. It will be explained to potential subjects/caregivers 
that receiving the practice cap does not require them to later enroll in the research study or to 
complete EEG tasks at a subsequent research visit. The practice caps are of negligible monetary 
value and are not believed to unduly influence subjects’ consideration of participating in the study. 
Compensation 
Subjects will receive $50 for each completed visit. If a participant cannot complete any study visits, 
they will be compensated at a rate of $15/hr. based on what they have completed. 
Payment will be in the form of a reloadable debit card (ClinCard). We will provide the card and load 
money onto the card after each completed visit. We will also administer a handout that will explain how 
to use it. 
Because this research study involves payment for participation, we are required by federal Internal 
Revenue Service (IRS) rules to collect and use participants’ social security or tax ID number (SSN) in 
order to track the amount of money that we pay. We will only use the participants’ SSN to keep track 
of how much money we pay to them and their SSN will not be used as part of this research. 
Families traveling significant distance to our clinic may be reimbursed for travel costs at the discretion 
of the PI. This may include mileage, hotel, airfare and/or other associated costs. 
11.0 Withdrawal of Subjects 
Any participant may be discontinued from the study at the discretion of the investigators if this is 
deemed to be in the best interest of the participant or if they are not compliant with study-specific 
requirements. A subject may voluntarily withdraw from the study at any time and for any reason. If a 
subject withdraws, at his or her request or at the request of his or her caregiver or legal representative, 
the reason(s) should be documented in the subject's source document. Any research information 
recorded for, or resulting from, participation in this research study prior to the date that the subject 
formally withdrew their consent will be retained and may continue to be used and disclosed by the 
investigators for research purposes; however, no new data will be collected. 
12.0 Risks to Subjects 
Confidentiality of records and results: The neurophysiological, medical, psychological, and behavioral 
measures will be made anonymous and labeled only by a unique identification number. Appropriate 
precautions will be taken to protect the confidentiality of all data. All subjects will be informed of this, 
including the HIPAA privacy of information act.  
Behavioral and cognitive testing: There are minimal risks anticipated to participating in observational, 
clinical behavioral assessments. In the rare case that an individual might become overly fatigued or 
distressed during the assessment session, the session will be discontinued and rescheduled. 

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Evaluation procedures are designed to be compatible with the attention span of a person with 
intellectual disability, greater activity level, and need for access to his or her parents or guardians. The 
NIH Cognitive Toolbox specifically created downward developmental extensions to accommodate for 
these needs. In addition, specialized procedures will be accompanied with specialized approaches 
such as using social stories and visual aids. There will be praise for task attention and overall 
emphasis on reducing language demands. The clinical investigators have extensive experience 
working clinically with developmentally disabled patients and their families. 
EEG: EEG testing is considered minimal risk. The most common risk associated with these tasks is 
boredom. The EEG nets do not use an electrode gel, but rather a saline solution and sponges which 
has a very low risk of skin irritation. The candidate and EEG technicians are highly experienced with 
doing EEG with sensitive populations such as children and subjects with neurodevelopmental 
disorders. 
13.0 Potential Benefits to Subjects 
Direct benefit to study participants is minimal. The major immediate benefit of participation in the 
proposed studies would be screening for symptoms of ASD and related disorders. Subjects may 
receive benefit from a complete evaluation including diagnostic, psychiatric, psychological, and 
medical evaluations in the form of a report. For individuals who are diagnosed with ASD or new 
diagnosis of FXS, appropriate referrals can be made if desired by study participants and their 
guardians. Caregivers and guardians may receive information regarding understanding the subject's 
behavior which may benefit them. We may also provide resources to connect them to local, regional, 
and national support organizations for FXS. Families and subjects may appreciate contributing to the 
discovery of innovative FXS treatments that will benefit other individuals. Healthy subjects often have 
various motivations and different rewards for participating in the study. Some subjects will be primarily 
motivated by the participant compensation. Others may want to participate to learn more about their 
skills or are personally committed to developmental disability research.  
14.0 Data Management and Confidentiality 
Data Storage and Sharing 
All records will be handled in a confidential manner. All hard copy data will be stored in code-labeled 
binders in locked storage facilities with limited access. Any electronic files containing patient identifiers 
will be either stored on a secure server or password protected database. Only the Principal Investigator 
and his designees will have access to hard-copy or electronic data. 
Unique numeric identifiers that do not include any PHI will be assigned to each participant. 
Identification numbers will code data entered for computer analysis, and the designated study staff will 
keep all names and code numbers in a password‐protected folder. EEG scans recordings will be 
anonymized by using only the participant study IDs and the date and time of scanning/recording and 
will be stored on a secure server.  
Sociodemographic information collected from this research may be utilized to create a Universal 
Identification Number (GUID) for the National Database for Autism Research (NDAR). Data that is 
collected from each research visit may be shared with NDAR. Before this data is sent to NDAR, 
information such as name and birthplace city will be removed and replaced with a GUID. 
Research data gathered as part of this study may be shared and provided to other investigators 
affiliated with the Neurobehavioral and Neurology Research Teams at CCHMC for the purpose of data 
sharing. If information is requested by an investigator, the PI must approve the request. If participants 
are enrolled in multiple or linked studies, their research data will be shared across studies to reduce 
participant burden and avoid duplication of procedures. All shared data will be de-identified, and 
results will be published as group data without the use of characteristics that would identify individual 
participants. 

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Quality Control 
At point-of-entry, data values will undergo consistency edits (e.g., ID validation, range verification, 
duplicate detection) and personnel will be required to correct errors. Data management staff will run 
logic error programs to check for accuracy and irregularities within and across data structures and 
within and across sites. Quality assurance checks will be conducted regularly by site personnel as well 
as by data management staff. Regularly scheduled, and as needed, communications between the 
study team, the neuroimaging team and PI will clarify any inconsistencies and ambiguities in the data. 
Data Analysis 
Power Analysis: Aim 1A: Based on differences in PPF in FXS vs. TDC in our resting state EEG data, 
we expect an effect size of approximately .8 to 1.1, similar to effect sizes in low gamma power and N1 
amplitudes in FXS10, 12, 90, 91. Aim 2: The effect size for WLI differences between structured and 
random conditions is approximately .853. The primary comparisons for both Aim 1A and Aim 2 will be 
between diagnostic groups, FXS vs. TDC and FXS vs ASD, Therefore, to be statistically conservative, 
the nominal alpha of .05 will be divided by 4, i.e.  .0125 per hypothesis. Using the available FXS vs. 
TDC effect size of 0.8, 40 subjects in each of the three diagnostic groups yields 84.3% power at a 
level of significance = .0125. Effects size data for FXS vs. ASD for these measures is not available but 
may be estimable for future studies. Aim 3: This aim is novel, without a strong basis for estimating an 
effect size. AAE effect size of PPF enhancement (vs. sham) in our preliminary data is between .89 and 
.97 (obtained peak frequencies of 9.3 to 9.6 Hz). For the within-groups crossover, an AAE trial to 
detect treatment differences in a 2 X 2 crossover design (20/group) yields approximately 80% power to 
identify group differences with an effect size of .9 at an alpha of 0.05. 
Statistical analysis: PPF (Aim 1A) and WLI (Aim 2) were selected as primary response variables 
since they will be individually optimized as starting parameters in Aim 3. Planned comparisons include 
FXS vs. TDC and FXS vs. ASD. FXS vs. TDC will be of primary interest, but confirmation of FXS vs. 
ASD can weaken the inference that differences in intellectual ability and genetic liability alone account 
for group difference. Covariates: FXS vs. TDC model will include age, sex, and non-verbal IQ. FXS vs. 
ASD will additionally include co-occurring psychiatric diagnoses, psychiatric medication(s) (4 
categories), and SRS score. A group by sex interaction will be added to each model given known sex 
differences in FXS15. Aim 1B: Murine data will consist of a parallel model to Aim 1A with three 
neurodynamic variables (PPF, event duration, and event number) and independent variable of group 
(KO or WT; 80 in each group). Aim 3: A linear model with repeated measures will test a 2-treatment, 
2-period, 2-sequence balanced-crossover design (Williams, 1989) with Kenward-Roger adjustment92 
applied. Further, as in Aim 1A and 2, two separate models will be examined for FXS vs. TDC and FXS 
vs. ASD. The responses of interest in Aim 3 are 40Hz ITC and WLI change (AAE vs. sham). The fixed 
effects of diagnostic group, treatment, the interaction between diagnostic group and treatment, 
treatment sequence, and period will be examined. Potential carryover effects will also be assessed. 
Covariates: Covariates for each comparison will be identical to models for Aim 1A and 2 above. The 
adjusted (least squares) means for the diagnostic group by treatment combination will allow to 
compare within and between diagnostic groups.  Within the FXS group, for example, the treatment 
effect will be by testing the contrast (FXS treatment – FXS sham) at the 0.05 level of significance.  
Exploratory analyses: Aim 1A: Other neurodynamic response variables (event rate and event 
duration) and chirp response (40Hz ITC and gamma ERSP) will be modeled as PPF. If the sex x 
diagnostic group interaction is significant (p < .05), subsequent post-hoc inter-diagnostic comparisons 
will be made for each sex. Lastly, for Aim 1A, the five variables representing neurodynamics and chirp 
response (PPF, event rate, event duration, 40 Hz ITC, and gamma ERSP) will be examined as a 
multivariate response across two paradigms (rest and chirp). As in the models above, diagnostic group 
will be the independent variable along with the appropriate covariates as described above. Aim 1C: To 
identify which FXS genotype (males with or without mosaicism and females) has similar alterations in 
neurodynamic variables as the Fmr1-/- KO (vs. WT) we will derive 95% confidence intervals for the 
effect sizes for each of the three neurodynamic variables for each of the three FXS subgroups (vs. 

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their respective matched controls) and compare to the confidence interval for the effect size based on 
the Fmr1-/- KO vs. WT comparison.  Lastly, significant differences between the FXS effect sizes and 
the murine effect size will be explored using bootstrap methodology where the resampling will be 
within each subgroup (human and mouse). 
15.0 Provisions to Monitor the Data to Ensure the Safety of Subjects 
There are no known risks or side effects associated with auditory entrainment; this stimulation 
is considered minimal risk. However, given the potential of neuromodulation and our site’s long 
history with TMS neuromodulation and clinical trials in FXS and ASD populations, we have opted to 
implement standardized safety measures and review study progress through a data safety monitoring 
board (DSMB).  
Dr. Sam Vaughn, Outpatient Clinical Director with the Division of Psychiatry at CCHMC, will serve as 
the DSMB Chair. Dr. Vaughn assumes this role for the Pedapati lab neurodevelopmental disorders 
interventional trials, and he is divisionally supported to execute this work. The monitor will review 
recruitment and adverse events every 6 months and report their assessment to the P.I. The 
independent monitor will also review any SAEs and significant unanticipated events as they occur. 
A physician will monitor data quality and adverse events at each visit. Any adverse events will be 
reported to the PI. All adverse events will be compiled, and reported in summary form to the IRB, on 
an annual basis and at the conclusion of the study. Any SAE during any study related activity 
(assessments, EEG, etc.) will be immediately reported to the DSMB and CCHMC IRB. In addition, the 
PI will review with the primary mentorship team if any future changes are deemed necessary to the 
study protocol to minimize future risk. 
16.0 Provisions to Protect the Privacy Interests of Subjects 
All subjects will be assured that any information collected about the participants as part of this study, 
including their identity, will be kept confidential. Data will be identified only by a unique identification 
number and not by the subject’s name. No information about the participant, or provided by the 
participant during the research, will be disclosed to others without his/her written permission, except if 
necessary to protect his/her rights or welfare; or if required by law. 
In reports of our findings from this study, data will only be presented as group averages that make no 
reference to individual participants. Participant information will not be used for any other purpose than 
this study unless specific informed consent is obtained beforehand. 
17.0 Economic Burden to Subjects 
Subjects will not incur any costs to participate in this study. 
18.0 Consent Process 
Consent will be obtained according to the following procedures prior to the start of any study 
procedures. For every subject under 18 years of age, their parent(s)/legal guardian(s) will be required 
to give voluntary written informed consent. Written assent will be obtained from subjects who are at 
least eleven years of age and cognitively able to give assent. Due to the age range for this study (5 to 
15), some children will not be able to sign an assent form. 
Informed consent procedures for the study will be conducted by research staff or investigators prior to 
enrollment. Legally authorized representatives together with participants initially will receive written 
materials and be given an oral explanation of the study. After an LAR has read the consent and 
expressed willingness to enroll in the study, the individual obtaining the informed consent will review 
any points about which the potential participant/LAR is unclear, and the participant/LAR be invited to 
ask questions. Participants and their LARs will be informed during their initial consent, and on several 
additional occasions throughout their experience in the study, that refusal to participate in the study 
will in no way influence or restrict treatment services at CCHMC, and the participant is free to withdraw 

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at any time with no negative consequences. One signed and witnessed copy of the consent and 
assent forms will be stored in secure storage and the participants and LARs will be given a copy of the 
signed forms. 
Consent may take place via in-person electronic consent (using REDCap for the eConsent) or over the 
phone or video call (via paper or REDCap for eConsent). REDCap is a secure web application for 
building and managing online surveys and databases. While REDCap can be used to collect virtually 
any type of data (including 21 CFR Part 22, FISMA, and HIPAA-compliant environments), it is 
specifically geared to support online or offline data capture for research studies and operations. The 
CCHMC REDCap team has templates available for research use for eConsenting which includes 
Consent to be a Research Subject and Authorization to Use or Disclose (Release) Health Information 
that Identifies Participant for a Research Study (HIPAA). The REDCap electronic consent format does 
not accommodate the current CCHMC formatting which includes headers with logos and stamps on 
each page and will therefore require some modifications. E-consenting can be conducted following 
CCHMC Clinical Research Procedure Number 41-1.4 Section 3.5.2. 
19.0 Process to Document Consent in Writing 
We will follow SOP guidelines for written documentation of consent (HRP-091).  
20.0 Setting 
All research procedures will take place at Cincinnati Children’s Hospital Medical Center.  
21.0 Resources Available 
We believe we have all the resources available to conduct this research study. Our recruitment goal is 
feasible considering the size of the target population, and we have sufficient amounts of time, facility 
space, medical resources, and trained staff to conduct and complete the study. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 

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